Manipulation of non-terminally differentiated cells using the notch pathway

ABSTRACT

The present invention is directed to methods for the expansion of non-terminally differentiated cells (&#34;precursor cells&#34;) using agonists of Notch function, by inhibiting the differentiation of the cells without inhibiting proliferation (mitotic activity) such that an expanded population of non-terminally differentiated cells is obtained. The cells are preferably stem or progenitor cells. These expanded cells can be used in cell replacement therapy to provide desired cell populations and help in the regeneration of diseased and/or injured tissues. The expanded cell populations can also be made recombinant and used for gene therapy, or can be used to supply functions associated with a particular precursor cell or its progeny cell.

This invention was made with government support under grant number NS26084 awarded by the National Institutes of Health. The government hascertain rights in the invention.

1. FIELD OF THE INVENTION

The present invention is directed to methods for the expansion ofnon-terminally differentiated cells ("precursor cells") using Notchreagents, by maintaining the differentiation state of the cells withoutinhibiting proliferation ("mitotic activity") such that an expandedpopulation of non-terminally differentiated cells is obtained. The cellsare preferably stem or progenitor cells. These expanded cells can beused in cell replacement therapy to repopulate lost cell populations andhelp in the regeneration of diseased and/or injured tissues. Theexpanded cell populations can also be made recombinant and used for genetherapy, or can be used to supply functions (e.g., expressed proteinproducts) associated with of a particular precursor cell or its progenycells.

2. BACKGROUND OF THE INVENTION

The developmental processes that govern the ontogeny of multicellularorganisms, including humans, depends on the interplay between signalingpathways, which gradually narrow the developmental potential of cellsfrom the original totipotent stem cell to the terminally differentiatedmature cell, which performs a specialized function, such as a heart cellor a nerve cell.

The fertilized egg is the cell from which all other cell lineagesderive, i.e., the ultimate stem cell. As development proceeds, earlyembryonic cells respond to growth and differentiation signals whichgradually narrow the cells' developmental potential, until the cellsreach developmental maturity, i.e., are terminally differentiated. Theseterminally differentiated cells have specialized functions andcharacteristics, and represent the last step in a multi-step process ofprecursor cell differentiation into a particular cell.

The transition from one step to the next in cell differentiation isgoverned by specific biochemical mechanisms which gradually control theprogression until maturity is reached. It is clear that thedifferentiation of tissues and cells is a gradual process which followsspecific steps until a terminally differentiated state is reached.

Gastrulation, the morphogenic movement of the early embryonic cell mass,results in the formation of three distinct germ cell layers, theectoderm, the mesoderm, and the endoderm. As cells in each germ celllayer respond to various developmental signals, specific organs aregenerated which are composed of specific differentiated cells. Forexample, the epidermis and the nervous system develop fromectoderm-derived cells, the respiratory system and the digestive tractare developed from endoderm-derived cells, and mesoderm-derived cellsdevelop into the connective tissues, the hematopoietic system, theurogenital system, muscle, and parts of most internal organs.

The following is a brief outline of how ectoderm, endoderm and mesodermare developed and further, how these three dermal layers give rise tothe different tissues of the body. For a general review of developmentsee Scott F. Gilbert, 1991, Developmental Biology, 3rd Edition, SinauerAssociates, Inc., Sunderland Mass.

The interaction between the dorsal mesoderm and the overlaying ectoderminitiates organogenesis. In this interaction the chordamesoderm directsthe ectoderm above it to form the neural tube which will eventually giverise to the brain and the spinal cord. The differentiation of the neuraltube into the various regions of the central nervous system is clear atthe gross anatomical level where morphogenetic changes shape specificconstrictions and bulges to form the chambers of the brain and thespinal cord. At the cellular level, cell migratory events rearrangevarious groups of cells. The neuroepithelial cells respond to growth anddifferentiation signals and eventually differentiate into the numeroustypes of neurons and supportive (glial) cells. Both neural tube andbrain are highly regionalized with each specific region serving distinctfunctional purposes (see FIG. 1). Each cell in this tissue has specificmorphological and biochemical characteristics. Differentiated cells arethe last step in a lineage where precursor cells responding todevelopmental cues progress to a more differentiated state until theyreach their terminal differentiation state. For example, ependymal cellswhich are the integral components of the neural tube lining can giverise to precursors which may differentiate into neurons or gliadepending on the developmental cues they will receive (Rakic et al.,1982, Neurosci. Rev. 20:429-611).

The neural crest derives from the ectoderm and is the cell mass fromwhich an extraordinary large and complex number of differentiated celltypes are produced. (see Table I), including the peripheral nervoussystem, pigment cells, adrenal medulla and certain areas of the headcartilage.

                                      TABLE I                                     __________________________________________________________________________    Major Neural Crest Derivatives*                                               Pigment                                                                              Sensory   Autonomic  Skeletal and                                                                             Skeletal and                           cells  nervous system                                                                          nervous system                                                                           connective tissue                                                                        connective tissue                      __________________________________________________________________________    TRUNK CREST (INCLUDING CERVICAL CREST)                                        Melanocytes                                                                          Spinal ganglia                                                                          Symphathetic                                                                             Mesenchyme of dorsal                                                                     Adrenal                                Xanthophores                                                                         Some contributions to                                                                   Superior cervical                                                                        fin in amphibia                                                                          medulla                                (erythrophores)                                                                      vagal (X) root ganglia                                                                  ganglion   Walls of aortic                                                                          Type I cells                           Iridophores      Prevertebral ganglia                                                                     arches     of carotid                             (guanophores)    Paravertebral ganglia                                                                    Connective tissue of                                                                     body                                   in dermis        Adrenal medulla                                                                          parathyroid                                                                              Parafollicle                           epidermis                              (calcitonin-                           and epidermal    Parasympahtetic       producing)                             derivates        Remark's ganglion     cells of                                                Pelvic plexus         thyroid                                                 Visceral and enteric                                                          ganglia                                                             Some supportive cells                                                         Glia (oligodendrocytes)                                                       Schwann sheath cells                                                          Some contribution to                                                          meninges                                                               CRANIAL CREST                                                                 Small, belated                                                                       Trigeminal (V)                                                                          Parasympahtetic ganglia                                                                  Most visceral                                     contribution                                                                         Facial (VII) root                                                                       Ciliary    cartilages                                               Glossopharyngeal (IX)                                                                   Ethmoid    Trabeculae carneae (ant.)                                root (superior                                                                          Sphenopalatine                                                                           Contributes cells to                                     ganglia)  Submandibular                                                                            posterior trabeculae,                                    Vagal (X) root (jugular                                                                            basal plate, para-                                       ganglia)             chordal cartilages                                                            Odontoblasts                                             Supportive cells     Head mesenchyme                                                               (membrane bones)                                  __________________________________________________________________________     *Derived from Gilbert, 1991, Developmental Biology, 3rd Edition, Sinauer      Associates, Inc., Sunderland MA, p. 182.                                 

The fate of neural crest cells will depend on where they migrate andsettle during development since the cells will encounter differentdifferentiation and growth signals that govern their ultimatedifferentiation. The pluripotentiality of neural crest cells is wellestablished (LeDouarin et al., 1975, Proc. Natl. Acad. Sci USA72:728-732). A single neural crest cell can differentiate into severaldifferent cell types. Transplantation experiments of cell populations orsingle neural crest cells point to the remarkably plasticdifferentiation potential of these cells. Even though the cell lineagesof the various differentiation pathways have not been established to thedegree they have in the hematopoietic development, the existence ofmulti-potential cell precursors, reminiscent to those seen in thehematopoietic system, is well founded.

The cells covering the embryo after neurulation form the presumptiveepidermis. The epidermis consists of several cellular layers whichdefine a differentiation lineage starting from the undifferentiated,mitotically active basal cells to the terminally differentiatednon-dividing keratinocytes. The latter cells are eventually shed andconstantly replenished by the underlying less differentiated precursors.Psoriasis, a pathogenic condition of the skin results from theexfoliation of abnormally high levels of epidermal cells.

Skin is not only the derivative of epidermis. Interactions betweenmesenchymal dermis, a tissue of mesodermal origin and the epidermis atspecific sites, result in the formation of cutaneous appendages, hairfollicles, sweat glands and apocrine glands. The cell ensemble thatproduces hairs is rather dynamic in that the first embryonic hairs areshed before birth and replaced by new follicles (vellus). Vellus, ashort and silky hair, remains on many parts of the body which areconsidered hairless, e.g., forehead and eye lids. In other areas velluscan give way to "terminal" hair. Terminal hair can revert into theproduction of unpigmented vellus, a situation found normally in malebaldness.

The endoderm is the source of the tissues that line two tubes within theadult body. The digestive tube extends throughout the length of thebody. The digestive tube gives rise not only to the digestive tract butalso to, for example, the liver, the gallbladder and the pancreas. Thesecond tube, the respiratory tube, forms the lungs and part of thepharynx. The pharynx gives rise to the tonsils, thyroid, thymus, andparathyroid glands.

The genesis of the mesoderm which has also been referred to as themesengenic process gives rise to a very large number of internal tissueswhich cover all the organs between the ectodermal wall and the digestiveand respiratory tubes. As is the case with all other organs it is theintricate interplay between various intercellular signaling events andthe response of non-terminally differentiated precursor cells that willeventually dictate specific cellular identities. To a large degree organformation depends on the interactions between mesenchymal cells with theadjacent epithelium. The interaction between dermis and epidermis toform, e.g., hairs, has been described above. The formation of the limbs,the gut organs, e.g., liver or pancreas, kidney, teeth, etc., all dependon interactions between specific mesenchymal and epithelial components.In fact, the differentiation of a given epithelium depends on the natureof the adjacent mesenchyme. For example, when lung bud epithelium iscultured alone, no differentiation occurs. However, when lung budepithelium is cultured with stomach mesenchyme or intestinal mesenchyme,the lung bud epithelium differentiates into gastric glands or villi,respectively. Further, if lung bud epithelium is cultured with livermesenchyme or bronchial mesenchyme, the epithelium differentiates intohepatic cords or branching bronchial buds, respectively.

2.1. ADULT TISSUES AND PRECURSOR CELLS

Embryonic development produces the fully formed organism. Themorphologic, i.e., cellular boundaries of each organ are defined and inthe juvenile or adult individual the maintenance of tissues whetherduring normal life or in response to injury and disease, depends on thereplenishing of the organs from precursor cells that are capable ofresponding to specific developmental signals.

The best known example of adult cell renewal via the differentiation ofimmature cells is the hematopoietic system. Here, developmentallyimmature precursors (hematopoietic stem and progenitor cells) respond tomolecular signals to gradually form the varied blood and lymphoid celltypes.

While the hematopoietic system is the best understood self renewingadult cellular system it is believed that most, perhaps all, adultorgans harbor precursor cells that under the right circumstances, can betriggered to replenish the adult tissue. For example, thepluripotentiality of neural crest cells has been described above. Theadult gut contains immature precursors which replenish thedifferentiated tissue. Liver has the capacity to regenerate because itcontains hepatic immature precursors; skin renews itself, etc. Throughthe mesengenic process, most mesodermal derivatives are continuouslyreplenished by the differentiation of precursors. Such repairrecapitulates the embryonic lineages and entails differentiation pathswhich involve pluripotent progenitor cells.

Mesenchymal progenitor cells are pluripotent cells that respond tospecific signals and adopt specific lineages. For example, in responseto bone morphogenic factors, mesenchymal progenitor cells adopt a boneforming lineage. For example, in response to injury, mesodermalprogenitor cells can migrate to the appropriate site, multiply and reactto local differentiation factors, consequently adopting a distinctdifferentiation path. It has been suggested that the reason that only alimited tissue repair is observed in adults is because there are too fewprogenitor cells which can adopt specific differentiation lineages. Itis clear that if such progenitor cells could be expanded, then thetissue repair could be much more efficient. An expanded pool of stem andprogenitor cells, as well as non-terminally differentiated cellssupplying a desired differentiation phenotype, would be of great valuein gene therapy and myriad therapeutic regimens.

2.2. THE NOTCH PATHWAY

Genetic and molecular studies have led to the identification of a groupof genes which define distinct elements of the Notch signaling pathway.While the identification of these various elements has come exclusivelyfrom Drosophila using genetic tools as the initial guide, subsequentanalyses have lead to the identification of homologous proteins invertebrate species including humans. FIG. 2 depicts the molecularrelationships between the known Notch pathway elements as well as theirsubcellular localization (Artavanis-Tsakonas et al., 1995, Science268:225-232).

The extracellular domain of Notch carries 36 EGF-like repeats, two ofwhich have been implicated in interactions with the Notch ligandsSerrate and Delta. Delta and Serrate are membrane bound ligands with EGFhomologous extracellular domains, which interact physically with Notchon adjacent cells to trigger signaling.

Functional analyses involving the expression of truncated forms of theNotch receptor have indicated that receptor activation depends on thesix cdc10/ankyrin repeats in the intracellular domain. Deltex andSuppressor of Hairless, whose over-expression results in an apparentactivation of the pathway, associate with those repeats.

Deltex is a cytoplasmic protein which contains a ring zinc finger.Suppressor of Hairless on the other hand, is the Drosophila homologue ofCBF1, a mammalian DNA binding protein involved in the Epstein-Barrvirus-induced immortalization of B cells. It has been demonstrated that,at least in cultured cells, Suppressor of Hairless associates with thecdc10/ankyrin repeats in the cytoplasm and translocates into the nucleusupon the interaction of the Notch receptor with its ligand Delta onadjacent cells (Fortini and Artavanis, 1994, Cell 79:273-282). Theassociation of Hairless, a novel nuclear protein, with Suppressor ofHairless has been documented using the yeast two hybrid systemtherefore, it is believed that the involvement of Suppressor of Hairlessin transcription is modulated by Hairless (Brou et al., 1994, Genes Dev.8:2491; Knust et al. 1992, Genetics 129:803).

Finally, it is known that Notch signaling results in the activation ofat least certain bHLH genes within the Enhancer of split complex(Delidakis et al., 1991, Genetics 129:803). Mastermind encodes a novelubiquitous nuclear protein whose relationship to Notch signaling remainsunclear but is involved in the Notch pathway as shown by geneticanalysis (Smoller et al., 1990, Genes Dev. 4:1688).

The generality of the Notch pathway manifests itself at differentlevels. At the genetic level, many mutations exist which affect thedevelopment of a very broad spectrum of cell types in Drosophila.Knockout mutations in mice are embryonic lethals consistent with afundamental role for Notch function (Swiatek et al., 1994, Genes Dev.8:707). Mutations in the Notch pathway in the hematopoietic system inhumans are associated with lymphoblastic leukemia (Ellison et al., 1991,Cell 66:649-661). Finally the expression of mutant forms of Notch indeveloping Xenopus embryos interferes profoundly with normal development(Coffman et al., 1993, Cell 73:659).

The expression patterns of Notch in the Drosophila embryo are complexand dynamic. The Notch protein is broadly expressed in the early embryo,and subsequently becomes restricted to uncommitted or proliferativegroups of cells as development proceeds. In the adult, expressionpersists in the regenerating tissues of the ovaries and testes (reviewedin Fortini et al., 1993, Cell 75:1245-1247; Jan et al., 1993, Proc.Natl. Acad. Sci. USA 90:8305-8307; Sternberg, 1993, Curr. Biol.3:763-765; Greenwald, 1994, Curr. Opin. Genet. Dev. 4:556-562;Artavanis-Tsakonas et al., 1995, Science 268:225-232). Studies of theexpression of Notch1, one of three known vertebrate homologues of Notch,in zebrafish and Xenopus, have shown that the general patterns aresimilar; with Notch expression associated in general with non-terminallydifferentiated, proliferative cell populations. Tissues with highexpression levels include the developing brain, eye and neural tube(Coffman et al., 1990, Science 249:1438-1441; Bierkamp et al., 1993,Mech. Dev. 43:87-100). While studies in mammals have shown theexpression of the corresponding Notch homologues to begin later indevelopment, the proteins are expressed in dynamic patterns in tissuesundergoing cell fate determination or rapid proliferation (Weinmaster etal., 1991, Development 113:199-205; Reaume et al., 1992, Dev. Biol.154:377-387; Stifani et al., 1992, Nature Genet. 2:119-127; Weinmasteret al., 1992, Development 116:931-941; Kopan et al., 1993, J. Cell Biol.121:631-641; Lardelli et al., 1993, Exp. Cell Res. 204:364-372; Lardelliet al., 1994, Mech. Dev. 46:123-136; Henrique et al., 1995, Nature375:787-790; Horvitz et al., 1991, Nature 351:535-541; Franco del Amo etal., 1992, Development 115:737-744). Among the tissues in whichmammalian Notch homologues are first expressed are the pre-somiticmesoderm and the developing neuroepithelium of the embryo. In thepre-somitic mesoderm, expression of Notch1 is seen in all of themigrated mesoderm, and a particularly dense band is seen at the anterioredge of pre-somitic mesoderm. This expression has been shown to decreaseonce the somites have formed, indicating a role for Notch in thedifferentiation of somatic precursor cells (Reaume et al., 1992, Dev.Biol. 154:377-387; Horvitz et al., 1991, Nature 351:535-541). Similarexpression patterns are seen for mouse Delta (Simske et al., 1995,Nature 375:142-145).

Within the developing mammalian nervous system, expression patterns ofNotch homologue have been shown to be prominent in particular regions ofthe ventricular zone of the spinal cord, as well as in components of theperipheral nervous system, in an overlapping but non-identical pattern.Notch expression in the nervous system appears to be limited to regionsof cellular proliferation, and is absent from nearby populations ofrecently differentiated cells (Weinmster et al., 1991, Development113:199-205; Reaume et al., 1992, Dev. Biol. 154:377-387; Weinmaster etal., 1992, Development 116:931-941; Kopan et al., 1993, J. Cell Biol.121:631-641; Lardelli et al., 1993, Exp. Cell Res. 204:364-372; Lardelliet al., 1994, Mech. Dev. 46:123-136; Henrique et al., 1995, Nature375:787-790; Horvitz et al., 1991, Nature 351:535-541). A rat Notchligand is also expressed within the developing spinal cord, in distinctbands of the ventricular zone that overlap with the expression domainsof the Notch genes. The spatio-temporal expression pattern of thisligand correlates well with the patterns of cells committing to spinalcord neuronal fates, which demonstrates the usefulness of Notch as amarker of populations of cells for neuronal fates (Henrique et al.,1995, Nature 375:787-790). This has also been suggested for vertebrateDelta homologues, whose expression domains also overlap with those ofNotch1 (Larsson et al., 1994, Genomics 24:253-258; Fortini et al., 1993,Nature 365:555-557; Simske et al., 1995, Nature 375:142-145). In thecases of the Xenopus and chicken homologues, Delta is actually expressedonly in scattered cells within the Notch1 expression domain, as would beexpected from the lateral specification model, and these patterns"foreshadow" future patterns of neuronal differentiation (Larsson etal., 1994, Genomics 24:253-258; Fortini et al., 1993, Nature365:555-557).

Other vertebrate studies of particular interest have focused on theexpression of Notch homologues in developing sensory structures,including the retina, hair follicles and tooth buds. In the case of theXenopus retina, Notch1 is expressed in the undifferentiated cells of thecentral marginal zone and central retina (Coffman et al., 1990, Science249:1439-1441; Mango et al., 1991, Nature 352:811-815). Studies in therat have also demonstrated an association of Notch1 with differentiatingcells in the developing retina have been interpreted to suggest thatNotch1 plays a role in successive cell fate choices in this tissue(Lyman et al., 1993, Proc. Natl. Acad. Sci. USA 90:10395-10399).

A detailed analysis of mouse Notch1 expression in the regeneratingmatrix cells of hair follicles was undertaken to examine the potentialparticipation of Notch proteins in epithelial/mesenchymal inductiveinteractions (Franco del Amo et al., 1992, Development 115:737-744).Such a role had originally been suggested for Notch1 based on the itsexpression in rat whiskers and tooth buds (Weinmaster et al., 1991,Development 113:199-205). Notch1 expression was instead found to belimited to subsets of non-mitotic, differentiating cells that are notsubject to epithelial/mesenchymal interactions, a finding that isconsistent with Notch expression elsewhere.

Expression studies of Notch proteins in human tissue and cell lines havealso been reported. The aberrant expression of a truncated Notch1 RNA inhuman T-cell leukemia results from a translocation with a breakpoint inNotch1 (Ellisen et al., 1991, Cell 66:649-661). A study of human Notch1expression during hematopoiesis has suggested a role for Notch1 in theearly differentiation of T-cell precursors (Mango et al., 1994,Development 120:2305-2315). Additional studies of human Notch1 andNotch2 expression have been performed on adult tissue sections includingboth normal and neoplastic cervical and colon tissue. Notch1 and Notch2appear to be expressed in overlapping patterns in differentiatingpopulations of cells within squamous epithelia of normal tissues thathave been examined and are clearly not expressed in normal columnarepithelia, except in some of the precursor cells. Both proteins areexpressed in neoplasias, in cases ranging from relatively benignsquamous metaplasias to cancerous invasive adenocarcinomas in whichcolumnar epithelia are replaced by these tumors (Mello et al., 1994,Cell 77:95-106).

Insight into the developmental role and the general nature of Notchsignaling has emerged from studies with truncated, constitutivelyactivated forms of Notch in several species. These recombinantlyengineered Notch forms, which lack extracellular ligand-binding domains,resemble the naturally occurring oncogenic variants of mammalian Notchproteins and are constitutively activated using phenotypic criteria(Greenwald, 1994, Curr. Opin. Genet. Dev. 4:556; Fortini et al., 1993,Nature 365:555-557; Coffman et al., 1993, Cell 73:659-671; Struhl etal., 1993, Cell 69:1073; Rebay et al., 1993, Genes Dev. 7:1949; Kopan etal., 1994, Development 120:2385; Roehl et al., 1993, Nature 364:632).

Ubiquitous expression of activated Notch in the Drosophila embryosuppresses neuroblast segregation without impairing epidermaldifferentiation (Struhl et al., 1993, Cell 69:331; Rebay et al., 1993,Genes Dev. 7:1949).

Persistent expression of activated Notch in developing imaginalepithelia likewise results in an overproduction of epidermis at theexpense of neural structures (Struhl et al., 1993, Cell 69:331).

Neuroblast segregation occurs in temporal waves that are delayed but notprevented by transient expression of activated Notch in the embryo(Struhl et al., 1993, Cell 69:331).

Transient expression in well-defined cells of the Drosophila eyeimaginal disc causes the cells to ignore their normal inductive cues andto adopt alternative cell fates (Fortini et al., 1993, Nature365:555-557).

Studies utilizing transient expression of activated Notch in either theDrosophila embryo or the eye disc indicate that once Notch signalingactivity has subsided, cells may recover and differentiate properly orrespond to later developmental cues (Fortini et al., 1993, Nature365:555-557; Struhl et al., 1993, Cell 69:331).

For a general review on the Notch pathway and Notch signaling, seeArtavanis-Tsakonas et al., 1995, Science 268:225-232.

Citation or identification of any reference in Section 2 or any othersection of this application shall not be construed as an admission thatsuch reference is available as prior art to the present is invention.

3. SUMMARY OF THE INVENTION

The present invention is directed to methods for the expansion ofnon-terminally differentiated cells ("precursor cells") by activatingthe Notch pathway in a precursor cell such that differentiation of theprecursor cell is inhibited without destroying the ability of the cellto proliferate. The precursor cell is preferably a stem or progenitorcell. The present invention is also directed to methods for theexpansion of precursor cells in precursor cell containing-populations byactivating the Notch pathway in the cells such that the differentiationof the stem cell is inhibited without affecting the mitotic activity ofthe stem cells. Further, the precursor cells can be isolated from a cellpopulation, if desired, before or after Notch pathway activation.Activation of the Notch pathway is preferably achieved by contacting thecell with a Notch ligand, e.g., in soluble form or recombinantlyexpressed on a cell surface or immobilized on a solid surface, or byintroducing into the cell a recombinant nucleic acid expressing adominant active Notch mutant or an activating Notch ligand, or othermolecule that activates the Notch pathway.

Activating Notch in the precursor cell renders the precursor cellrefractory to differentiation signals, thus substantially inhibitingdifferentiation and allowing maintenance of the cell in itsdifferentiation stage, and, optionally, expansion of the cell uponexposure to cell growth conditions. Thus, the methods of the inventionprovide precursor cells of a specific differentiation state. Thus, inone embodiment, such a cell which expresses a desired differentiationphenotype (e.g., production of a desired hormone or growth factor) canis be administered to a patient wherein the differentiation phenotype istherapeutically useful (e.g., hormone or growth factor deficiency).Alternatively, an expanded stem or progenitor cell population producedby activation of Notch and cell growth can be used to replace orsupplement the stem or progenitor cell lineage in a patient byadministration of such cell population. If desired, members of theexpanded cell population can be induced to differentiate in vitro priorto in vivo administration, so as to supply to the patient the functionof a more differentiated cell population. Preferably, the Notchactivation is carried out in vitro and is reversible so that upon invivo administration of the cells differentiation can occur. Thus, forexample, in a preferred embodiment, a Notch ligand is used to activateNotch on the cells, e.g., by being added in soluble form to the cellmedia, or contacting the cells with a layer of cells in cultureexpressing the Notch ligand (e.g., Delta, Serrate) on its surface.

The precursor cells to be expanded in the present invention can beisolated from a variety of sources using methods known to one skilled inthe art (see Section 5.5, infra). The precursor cells can be of anyanimal, preferably mammalian, most preferably human, and can be ofprimary tissue, cell lines, etc. The precursor cells can be ofectodermal, mesodermal or endodermal origin. Any precursor cells whichcan be obtained and maintained in vitro can potentially be used inaccordance with the present invention. In a preferred embodiment, theprecursor cell is a stem cell. Such stem cells include but are notlimited to hematopoietic stem cells (HSC), stem cells of epithelialtissues such as the skin and the lining of the gut, embryonic heartmuscle cells, and neural stem cells (Stemple and Anderson, 1992, Cell71:973-985). The stem cells can be expanded under cell growthconditions, i.e., conditions that promote proliferation ("mitoticactivity") of the cells.

The least differentiated cell in a cell lineage is termed a stem cell.However, stem cell is an operational term. The classic definition of thestem cell is a cell which can divide to produce another stem cell(self-renewal capacity), as well as a cell which can differentiate alongmultiple specific differentiation paths. It is often the case that aparticular cell within a differentiation lineage, has derived from a"less" differentiated parent and can still divide and give rise to a"more" differentiated cellular progeny. FIG. 3 describesdiagrammatically hematopoietic development. Totipotent, pluripotent andprogenitor stem cells are referred to in the figure.

A "precursor cell" may or may not divide and can be triggered to adopt adifferent differentiation state but not necessarily a fullydifferentiated state, by responding to specific developmental signals.

The present invention is also directed to methods for use of theexpanded precursor cells for use in gene therapy as well as for use inproviding desired cell populations, e.g., for regenerating injuredand/or diseased tissues. The expanded precursor cell populations can beadministered to a patient using methods commonly known to those skilledin the art (see Section 5.8, infra). In other specific embodiments,after Notch activation and expansion, the precursor can be induced todifferentiate in vivo, or alternatively in vitro, followed byadministration to an individual, to provide a differentiated phenotypeto a patient. Additionally, Notch activation and expansion can becarried out in vitro subsequent to in vitro production of a precursorcell of a desired phenotype from a stem or progenitor cell.

The present invention is also directed to precursor cells containingrecombinant genes, such that the gene is inheritable and expressible bythe precursor cell or its progeny. These recombinant precursor cells canbe transplanted into a patient such that the desired gene is expressedin the patient to alleviate a disease state caused by the lack of ordeficient expression of the recombinant gene. The precursor cells can bemade recombinant either before or after precursor cell expansion.Methods of tranfecting the nucleic acid encoding the desired geneproduct such that the precursor cell or its progeny stably expresses thegene product are known to those of skill in the art and are describedinfra.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram showing regional specialization during human braindevelopment. (Gilbert, 1991, Developmental Biology, 3rd Edition, SinauerAssociates, Inc., Sunderland Mass., p. 166.)

FIG. 2 is a schematic diagram of the Notch signaling pathway. The Notchreceptor can bind to either Delta or Serrate through its extracellulardomain. Ligand binding can result in receptor multimerization that isstabilized by interactions between the intracellular ankyrin repeats ofNotch and the cytoplasmic protein Deltex. These events can control thenuclear translocation of the DNA-binding protein Suppressor of Hairlessand its known association with the Hairless protein. The transcriptionalinduction of the Enhancer of Split bHLH genes appears to depend on Notchsignaling.

FIG. 3 is a schematic diagram of the origin of mammalian blood andlymphoid cells. (Gilbert, 1991, Developmental Biology, 3rd Edition,Sinauer Associates, Inc., Sunderland Mass., p. 232).

FIGS. 4A-4D show the highly conserved ankyrin repeat region of Notch ,hum N (SEQ ID NO.:1), Tan-1 (SEQ ID NO.:2), Xen N (SEQ ID NO.:3), Dros N(SEQ ID NO.:4).

FIGS. 5A-F. Expression of activated Notch and neural differentiation incone cell precursors of transgenic flies bearing both sev-Notch^(nucl)and the activated Raf construct sE-raf^(torY9). Third-instar larval eyeimaginal discs were reacted with mouse monoclonal antibody C17.9C6directed against the intracellular domain of Notch and rat monoclonalantibody 7E8A10 directed against the neural antigen ELAV and visualizedwith immunofluorescent secondary antibodies using confocal microscopy.Low (5A-C) and high (5E-F) magnification images of posterior eye discregions showing nuclear Notch staining (green) in sevenless-expressingcells (5A,D), nuclei expressing ELAV (red) undergoing neuraldifferentiation (5B,E) and corresponding image overlays of both stainingpatterns (5C,F). The field shown in (5A-C) spans ommatidial rows 10-23,with the posterior margin of the disc visible at the left; nuclei thatexpress Notch protein do not express ELAV. Individual cone cellprecursor nuclei of similar developmental ages are labeled `N` in (5D)if they stain for Notch but not ELAV, and are labelled `E` in (5E) ifthey stain for ELAV but not Notch. Faint ELAV staining (red) was oftenobserved beneath strongly Notch-positive (green) cone cell precursornuclei; examples are indicated by asterisks in (5E). Optical sectioningrevealed that this ELAV staining corresponds to R1,3, 4, 6 and 7photoreceptor cell precursor nuclei that are located immediately belowand partially intercalated with the cone cell precursor nuclei.Identical staining patterns were observed for sev-Notch^(nucl) fliesbearing the activated Sevenless tyrosine kinase construct sev-S11 or theactivated Ras1 construct sevRas1^(Va112) instead of sE-^(torY9) (datanot shown).

FIGS. 6A-B. Co-expression of activated Notch and activated Sevenlessproteins in cone cell precursors of sevenless^(d2) flies bearingsev-Notch^(nucl) and sev-S11. Third-instar larval eye imaginal discswere reacted with rat polyclonal antibody Rat5 directed against theintracellular domain of Notch and mouse monoclonal antibody sev150C3directed against the 60 kD subunit of Sevenless and visualized withimmunofluorescent secondary antibodies using confocal microscopy. Thesevenless^(d2) allele produces no protein recognized by mAb sev150C3.(6A) Image overlay of two horizontal optical sections collected atslightly different apical levels within the same posterior eye discquadrant, showing expression of activated Notch (green) in most of thecone cell precursor nuclei and expression of activated Sevenless(purple) in most of the corresponding apical membranes of the cone cellprecursor population. The ring-shaped distribution of Sevenless proteinin each assembling ommatidium represents the apical microvillar tufts ofup to four cone cell precursors and the R7 precursor cell. (6B) Highermagnification image overlay similar to that in (6A), showing adeveloping ommatidium in which all four cone cell precursor nucleiexpress Notch (labelled `N`) and all or most cone cell precursor apicalmembrane tufts exhibit strong Sevenless expression (labelled `Sev`).

FIG. 7. Schematic representation of the epistatic relationship betweenNotch activation and the signalling pathway involving the sevenlessreceptor tyrosine kinase, Ras1 and Raf during neural induction of the R7cell precursor in Drosophila. Sevenless protein (Sev) in the R7 cellprecursor is activated by binding to its ligand Bride of sevenless(Boss), presented by the adjacent R8 cell, resulting in Ras1 activationpresumably via regulation of the activities of its guanine nucleotideexchange factor Son-of-sevenless and its GTPase-activating protein Gap1.Ras1 activation leads to the activation of Raf. This signalling pathwayis inhibited by Notch activation at some point downstream of Raf.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to methods for the expansion ofnon-terminally differentiated cells ("precursor cells") by activatingthe Notch pathway in a precursor cell such that the differentiation ofthe precursor cell is inhibited without destroying the ability of thecell to proliferate. As used herein, "precursor cells" shall mean anynon-terminally differentiated cells. The precursor cell is preferably astem or progenitor cell. The present invention is also directed tomethods for the expansion of precursor cells in precursor cellcontaining-populations by activating the Notch pathway in the cells suchthat the differentiation of the stem cell is inhibited without affectingthe mitotic activity of the cells. Further, the precursor cells can beisolated from a cell population, if desired, before or after Notchpathway activation. Activation of Notch pathway is preferably achievedby contacting the cell with a Notch ligand, e.g., in soluble form orrecombinantly expressed on a cell surface or immobilized on a solidsurface, or by introducing into the cell a recombinant nucleic acidexpressing a dominant active Notch mutant or an activating Notch ligand,or other molecule that activates the Notch pathway.

Agonists of the Notch pathway are able to activate the Notch pathway atthe level of protein-protein interaction or protein-DNA interaction.Agonists of Notch include but are not limited to proteins andderivatives comprising the portions of toporythmic proteins such asDelta or Serrate or Jagged (Lindsell et a., 1995, Cell 80:909-917) thatmediate binding to Notch, and nucleic acids encoding the foregoing(which can be administered to express their encoded products in vivo).In a preferred embodiment, the agonist is a protein or derivative orfragment thereof comprising a functionally active fragment such as afragment of a Notch ligand that mediates binding to a Notch protein. Inanother preferred embodiment, the agonist is a human protein or portionthereof (e.g., human Delta). In another preferred embodiment the agonistis Deltex or Suppressor of Hairless or a nucleic acid encoding theforegoing (which can be administered to express its encoded product invivo).

The Notch pathway is a signal transducing pathway comprising elementswhich interact, genetically and/or molecularly, with the Notch receptorprotein. For example, elements which interact with the Notch protein onboth a molecular and genetic basis are, for example, and not by way oflimitation, Delta, Serrate and Deltex. Elements which interact with theNotch protein genetically are, for example, and not by way oflimitation, Mastermind, Hairless and Suppressor of Hairless.

Activating Notch function in the precursor cell renders the precursorcell refractory to differentiation signals, thus substantiallyinhibiting differentiation and allowing maintenance of the cell in itsdifferentiation stage, and, optionally, expansion of the cell uponexposure to cell growth conditions. Thus, the methods of the inventionprovide precursor cells of a specific differentiation state. Thus, inone embodiment, such a cell which expresses a desired differentiationphenotype (e.g., production of a desired hormone or growth factor) canbe administered to a patient wherein the differentiation phenotype istherapeutically useful (e.g., hormone or growth factor deficiency).Alternatively, an expanded stem or progenitor cell population producedby activation of Notch and cell growth can be used to replace orsupplement the stem or progenitor cell lineage in a patient byadministration of such cell population. If desired, members of theexpanded cell population can be induced to differentiate in vitro priorto in vivo administration, so as to supply to the patient the functionof a more differentiated cell population. Preferably, the Notchactivation is carried out in vitro and is reversible so that upon invivo administration of the cells differentiation can occur. Thus, forexample, in a preferred embodiment, a Notch ligand is used to activateNotch on the cells, e.g., by being added in soluble form to the cellmedia, or contacting the cells with a layer of cells in cultureexpressing the Notch ligand (e.g., Delta, Serrate) on its surface.

The precursor cells to be expanded in the present invention can beisolated from a variety of sources using methods known to one skilled inthe art (see Section 5.5, infra). The precursor cells can be ofectodermal, mesodermal or endodermal origin. Any precursor cells whichcan be obtained and maintained in vitro can potentially be used inaccordance with the present invention. In a preferred embodiment, theprecursor cell is a stem cell. Such stem cells include but are notlimited to hematopoietic stem cells (HSC), stem cells of epithelialtissues such as the skin and the lining of the gut, embryonic heartmuscle cells, and neural stem cells (Stemple and Anderson, 1992, Cell71:973-985). The stem cells can be expanded under cell growthconditions, i.e., conditions that promote proliferation ("mitoticactivity") of the cells.

The least differentiated cell in a cell lineage is termed a stem cell.However, stem cell is an operational term. The classic definition of thestem cell is a cell which can divide to produce another stem cell(self-renewal capacity), as well as a cell which can differentiate alongmultiple specific differentiation paths. It is often the case that aparticular cell within a differentiation lineage, has derived from a"less" differentiated parent and can still divide and give rise to a"more" differentiated cellular progeny. FIG. 3 describesdiagrammatically hematopoietic development. Totipotent, pluripotent andprogenitor stem cells are referred to in the figure.

A "precursor cell" has specific biochemical properties, may or may notdivide and can be triggered to adopt a different differentiation statebut not necessarily a fully differentiated state, by responding tospecific developmental signals.

The present invention is also directed to methods for use of theexpanded precursor cells for use in gene therapy as well as for use inproviding desired cell populations and for use in regenerating injuredand/or diseased tissues. The expanded precursor cell populations can beadministered to a patient using methods commonly known to those skilledin the art (see Section 5.8, infra). In other specific embodiments,after Notch activation and expansion, the precursor cell can be inducedto differentiate in vivo, or alternatively in vitro, followed byadministration to an individual, to provide a differentiated phenotypeto a patient. Additionally, Notch activation and expansion can becarried out in vitro subsequent to in vitro production of a precursorcell of a desired phenotype from a stem or progenitor cell.

The present invention is also directed to precursor cells expressingrecombinant genes, such that the precursor cells express a desired gene.These recombinant precursor cells can be transplanted into a patientsuch that the desired gene is expressed in the patient to alleviate adisease state caused by the lack of expression of the recombinant gene.The precursor cells can be made recombinant either before or afterprecursor cell expansion. Methods of tranfecting the nucleic acidencoding the desired gene product such that the precursor cell or itsprogeny stably expresses the gene product are known to those of skill inthe art and are described infra.

The subject into which the expanded cells or their progeny areintroduced, or from which precursor cells can be derived, is preferablyan animal, including but not limited to animals such as cows, pigs,horses, chickens, cats, dogs, etc., and is preferably a mammal, and mostpreferably human.

In an embodiment of the present invention, the subjects to which thecells are administered are immunocompromised or immunosuppressed or havean immune deficiency. For example, the subject has Acquired ImmuneDeficiency Syndrome or has been exposed to radiation or chemotherapyregimens for the treatment of cancer, and the subjects are administeredhematopoietic or immune precursor cells such that the administered cellsperform a needed immune or hematopoietic function.

Preferably, the expanded precursor cell is originally derived from thesubject to which it is administered, i.e., the transplant is autologous.

For clarity of disclosure, and by way of limitation, the detaileddescription of the invention is divided into the following sub-sections:

(i) Notch signaling and stem cell differentiation,

(ii) Notch activation inhibits the differentiation of stem cells,

(iii) Activation of the Notch pathway,

(iv) Notch and terminal differentiation,

(v) Obtaining precursor cells,

(vi) Gene therapy,

(vii) Pharmaceutical compositions,

(viii) Transplantation.

5.1. NOTCH SIGNALING AND STEM CELL DIFFERENTIATION

The progression of a precursor cell to a more mature or differentiatedstate depends on a combination of signals that ultimately govern thedifferentiation steps. Specific factors, for example, bone morphogenicfactors or the various factors known to be important in hematopoiesis,for example, interleukin-5 or thrombopoietin, together withintercellular and cell-extracellular matrix interactions contribute tothe differentiation of a precursor cell along a specific differentiationpath.

The effectors of such contributions are the various signaling pathwayswhich transmit the extracellular signal to the nucleus, ultimatelychanging transcriptional expression patterns, i.e., genes expressed onlyin the tissue that is the cells' ultimate fate are switched on andconversely others are switched off, such that, e.g., kidney cellsexpress kidney-specific genes and do not express liver cell-specificproteins. In order for a precursor cell to respond to the variousextracellular signals, it must be competent to do so, for example, inorder to respond to a soluble factor the cell must express a receptorwhich can recognize the factor. Tissue competence has been articulatedin the classic studies of Waddington, 1940, Organisers and Genes,Cambridge University Press, Cambridge, England.

The present invention is based, at least in part, on the discovery thatthe Notch signaling pathway is not a pathway that transmits specificdevelopmental signals such that cell differentiation is effected, butrather it controls the competence of a precursor cell to interpret andrespond to differentiation signals. The Notch pathway is a general andevolutionarily conserved developmental "switch." Specifically, when theNotch pathway is activated in precursor cells, the precursor cells areunable to respond to particular differentiation signals but generallythe mitotic ability of the precursor cells remains (i.e., the cells canproliferate). The existence of the Notch pathway allows for themanipulation of the differentiation state of precursor cells withoutknowing all of the differentiation signals, e.g., growth factors, whichare required for the maintenance of a particular differentiation stateor for advancing the cell to a more differentiated state. In a preferredaspect, the inhibitory effect on differentiation by activating the Notchpathway with a Notch function agonist can be reversed by adding anantagonist of the Notch pathway or diluting out the Notch pathwayagonist.

5.2. NOTCH ACTIVATION INHIBITS THE DIFFERENTIATION OF PRECURSOR CELLS

Notch regulates the competence of many different cell types to respondto more specific signals, with the particular cell fates chosendepending upon the developmental history of each cell type and thespecific signaling pathways operating within it. When Notch function isactivated in a precursor cell (e.g., progenitor or stem cell), theprecursor cell can be prevented from differentiating even in thepresence of the correct differentiation signals. Once, however, Notchfunction activation subsides, the cells can respond again todevelopmental cues. We have shown, using human keratinocytes which havebeen transfected with activated forms of Notch, that while cells stablyexpressing activated Notch forms are prevented from differentiating,their proliferation potential is not affected.

The modulation of Notch pathway activity offers a novel and unique toolto manipulate the fate of precursor cells. A precursor at a givendevelopmental state can be "frozen" into that state by virtue ofactivating the Notch pathway. Importantly, these cells may be expanded,since Notch signaling activity may not destroy or, preferably, does notsubstantially impair, their ability to divide. Thus, precursor cells maybe expanded, ex vivo in order to provide a source of precursors whichare useful in gene therapy as well as tissue repair. Notch agonists arealso useful in cases where it is important to maintain a cell in aparticular differentiation state in order to provide indefinitely, orfor a given period of time, a chemical produced by a cell of thatdifferentiated state, to a particular tissue. In this latter embodiment,for example, it may be desired to activate Notch in the cellsadministered in vivo for a long period of time (e.g., hours or days) orsubstantially irreversibly, e.g., by excapsulating the cells with asoluble Notch agonist, or having them recombinantly express a Notchdominant active mutant from a constitutive promoter, respectively.

An embodiment of the present invention is to treat the desired cellpopulation with agonists of the Notch pathway and then either allowthese cells to proliferate in culture before transplanting them backinto the appropriate region, or directly transplanting them withoutnecessarily allowing them to proliferate in vitro. Antagonists can beused to reverse or neutralize the action of the Notch function agonist.For example, and not by way of limitation, a Notch ligand or a moleculethat mimics the ligand can be used to keep Notch receptor expressingcells in an "activated" state while withdrawal of the ligand willreverse that effect.

It is possible in many cases that the simple activation of Notch may notsuffice to expand the stem cells ex vivo. Subjecting the cell to growthconditions, e.g., culturing it in the presence of specific growthfactors or combinations of growth factors may be necessary,nevertheless, the importance of Notch pathway activation in these eventswill be essential since the presence of only those factors willgenerally not be sufficient to maintain those cells in culture withoutdifferentiation occurring.

5.3. ACTIVATION OF NOTCH FUNCTION

An agonist of Notch function is an agent that promotes activation ofNotch function. As used herein, "Notch function" shall mean a functionmediated by the Notch signaling pathway.

Notch function activation is preferably carried out by contacting aprecursor cell with a Notch function agonist. The agonist of Notchfunction can be a soluble molecule, recombinantly expressed as acell-surface molecule, on a cell monolayer with which the precursorcells are contacted, a molecule immobilized on a solid phase. In anotherembodiment, the Notch agonist can be recombinantly expressed from anucleic acid introduced into the precursor cells. Notch functionagonists of the present invention include Notch proteins and analogs andderivatives (including fragments) thereof; proteins that are otherelements of the Notch pathway and analogs and derivatives (includingfragments) thereof; antibodies thereto and fragments or otherderivatives of such antibodies containing the binding region thereof;nucleic acids encoding the proteins and derivatives or analogs; as wellas toporythmic proteins and derivatives and analogs thereof which bindto or otherwise interact with Notch proteins or other proteins in theNotch pathway such that Notch function is promoted. Such agonistsinclude but are not limited to Notch proteins and derivatives thereofcomprising the intracellular domain, Notch nucleic acids encoding theforegoing, and proteins comprising toporythmic protein domains thatinteract with Notch (e.g., the extracellular domain of Delta, Serrate orJagged). Other agonists include Deltex and Suppressor of Hairless. Theseproteins, fragments and derivatives thereof can be recombinantlyexpressed and isolated or can be chemically synthesized.

In a preferred embodiment the agonist is a protein consisting of atleast a fragment (termed herein "adhesive fragment") of the proteinsencoded by toporythmic genes which mediate binding to Notch proteins oradhesive fragments thereof. Toporythmic genes, as used herein, shallmean the genes Notch, Delta, Serrate, Jagged, Suppressor of Hairless andDeltex, as well as other members of the Delta/Serrate/Jagged family orDeltex family which may be identified by virtue of sequence homology orgenetic interaction and more generally, members of the "Notch cascade"or the "Notch group" of genes, which are identified by molecularinteractions (e.g., binding in vitro, or genetic interactions (asdepicted phenotypically, e.g., in Drosophila).

Vertebrate homologs of Notch pathway elements have been cloned andsequenced. For example, these include Serrate (Lindsell et al., 1995,Cell 80:909-917); Delta (Chitnis et al., 1995, Nature 375:761; Henriqueet al., 1995, Nature 375:787-790; Bettenhausen et al., 1995, Development121:2407); and Notch (Coffman et al., 1990, Science 249:1438-1441;Bierkamp et al., 1993, Mech. Dev. 43:87-100; Stifani et al., 1992,Nature Genet. 2:119-127; Lardelli et al., 1993, Exp. Cell Res.204:364-372; Lardelli et al., 1994, Mech. Dev. 46:123-136; Larsson etal., 1994, Genomics 24:253-258; Ellisen et al., 1991, Cell 66:649-661;Weinmaster et al., 1991, Development 113:199-205; Reaume et al., 1992,Dev. Biol. 154:377-387; Weinmster et al., 1992, Development 116:931-941;Franco del Amo et al., 1993, Genomics 15:259-264; and Kopan et al.,1993, J. Cell. Biol. 121:631-641).

In one embodiment, the Notch agonist is expressed from a recombinantnucleic acid. For example, in vivo expression of truncated, "activated"forms of the Notch receptor lacking the extra cellular, ligand bindingdomain results in gain of function mutant phenotypes. When analyzed atthe single cell level, these phenotypes demonstrate that expression ofsuch molecules in progenitor or stem cells, prevents the cells fromresponding to differentiation signals, thus inhibiting differentiation.It has also been mentioned that this process may be desired to bereversible, since when the activated Notch receptor is no longerexpressed the undifferentiated stem cells can respond to differentiationsignals and differentiate. Thus, preferably the Notch dominant activemutant is expressed inside the precursor cells from an induciblepromoter, such that expression can be induced In vitro for expansion,with the inducer lacking in vivo so that differentiation occurs afteradministration of the transplanted cells.

Alternatively, in another embodiment the agonist of Notch function isnot a recombinant dominant Notch active mutant.

Alternatively, in another embodiment, contacting of the precursor cellswith a Notch agonist is not done by incubation with other cellsrecombinantly expressing a Notch ligand on the cell surface (although inother embodiments, this method can be used).

In another embodiment, the recombinantly expressed Notch agonist is achimeric Notch protein which comprises the intracellular domain of Notchand the extracellular domain of another ligand-binding surface receptor.For example, a chimeric Notch protein comprising the EGF receptorextracellular domain and the Notch intracellular domain is expressed ina precursor cell. However, the Notch pathway will not be active unlessthe EGF receptor ligand EGF is contacted with the precursorcell-expressing the chimera. As with the inducible promoter controllingthe expression of the truncated form of Notch, the activity of thechimeric Notch protein is reversible; when EGF is removed from thecells, Notch activity will cease and the cell can then differentiate.Notch activity can again be turned on with the addition of the ligand.

A systematic deletion analysis of the intracellular domain of Notchdemonstrates that the Notch sequences that are both necessary andsufficient for the downstream signaling of the Notch receptor areconfined to the ankyrin repeats of the intracellular region (Matsuno etal., 1995, Development 121:2633-2644 and unpublished results). Using theyeast two hybrid system it was discovered that the ankyrin repeatsinteract homotypically.

Expression of appropriate deletion constructs in the defined cellularenvironment of the developing Drosophila eye demonstrates thatexpression of a polypeptide fragment comprising just the ankyrin repeatsresulted in an activated phenotype. Not surprisingly this is the part ofthe Notch protein which is most highly conserved among various species.FIGS. 4A-4D show the high sequence homology of ankyrin repeats acrossevolution.

These findings suggest that any small molecules, for example, but not byway of limitation, polypeptides or antibodies which bind to the Notchankyrin repeats, can block its function, and hence behave as antagonistsof the pathway. Conversely, molecules that mimic the Notch ankyrinrepeat activity can behave as agonists of the Notch pathway. Since theexpression of truncated forms of Notch give mutant phenotypes in thedeveloping Drosophila eye, genetic screens for modifiers of thesephenotypes can be used for identifying and isolating additional geneproducts that can act as agonists or antagonists of the pathway.

Genes that act as enhancers of the activated phenotypes are potentialagonists and those that act as suppressors are potential antagonists.

Deltex and Suppressor of Hairless are also agonists of Notch functionthat can be used. It has been shown that the activation of the Notchpathway, as judged by the induction of activated phenotypes similar tothose induced by the expression of activated forms of Notch, can beachieved by manipulating the expression of Deltex (Schweisguth andPosakony, 1994, Development 120:1477), as well as Suppressor of Hairless(Matsuno et al., 1995, Development 121:2633) both of which can interactwith the ankyrin repeats of Notch.

Using the yeast `interaction trap` assay (Zervos et al., 1993, Cell72:223-232), as well as cell culture co-localization studies, theprotein regions responsible for heterotypic interactions between Deltexand the intracellular domain of Notch, as well as homotypic interactionamong Deltex molecules were defined. The function of the Deltex-Notchinteraction domains was examined by in vivo expression studies. Takentogether, data from over-expression of Deltex fragments and from studiesof physical interactions between Deltex and Notch demonstrate thatDeltex positively regulates the Notch pathway through interactions withthe Notch ankyrin repeats.

Experiments involving cell cultures indicate that the Deltex-Notchinteraction prevents the cytoplasmic retention of Suppressor of Hairlessprotein, which is normally sequestered in the cytoplasm via associationwith the Notch ankyrin repeats and translocates to the nucleus whenNotch binds to its ligand, Delta. On the basis of these findings Deltexappears to regulate Notch activity by antagonizing the interactionbetween Notch and Suppressor of Hairless. The translocation of thenormally cytoplasmic Suppressor of Hairless protein to the nucleus whenNotch binds to a ligand (Fortini and Artavanis-Tsakonas, 1994, Cell79:273-282) is a convenient assay to monitor for Notch function as wellas for the ability of Notch agonists of the present invention toactivate Notch function.

Suppressor of Hairless has been shown to be a DNA binding protein.Genetic and molecular data indicate that the activity of Suppressor ofHairless can be influenced by its binding to the nuclear proteinHairless. Moreover it appears that the transcription of at least some ofthe bHLH genes of the Enhancer of split complex depends directly onNotch signaling and the ability of Suppressor of Hairless to recognizethe appropriate binding sites upstream of these genes. Manipulation ofthese various interactions (e.g., disrupting the interaction betweenNotch and Suppressor of Hairless with an antibody directed against theankyrin repeats) will result in modulating the activity of the Notchpathway.

Finally, the Notch pathway can be manipulated by the binding of Notchligands (e.g., Delta, Serrate) to the extracellular portion of the Notchreceptor. Notch signaling appears to be triggered by the physicalinteraction between the extracellular domains of Notch and itsmembrane-bound ligands on adjacent cells. The expression of full lengthligands on one cell triggers the activation of the pathway in theneighboring cell which expresses the Notch receptor. Not surprisingly,the ligands act as agonists of the pathway. On the other hand, theexpression of truncated Delta or Serrate molecules which lackintracellular domains expressed in neighboring cells results innon-autonomous, dominant negative phenotypes. This demonstrates thatthese mutant forms of the receptor act as antagonists of the pathway.

The definition of the various molecular interactions among the Notchpathway elements provides additional specific pharmacological targetsand assays which can be used to screen for Notch function agonists andantagonists. Having evaluated the consequences of a particular molecularmanipulation in vivo, this information can be used to design biochemicalin vitro screening assays for biological or pharmaceuticals thatinterfere or enhance Notch function.

Screening for molecules that will trigger the dissociation of the Notchankyrin repeats with Suppressor of Hairless and the subsequenttranslocation of Suppressor of Hairless from the cytoplasm to thenucleus results in the identification of agonists. The activation oftranscription of a reporter gene which has been engineered to carryseveral Suppressor of Hairless binding sites at its 5' end in a cellthat expresses Notch also results in the identification of agonists ofthe pathway.

Reversing the underlying logic of these assays leads to theidentification of antagonists. For example, cell lines expressing theaforementioned reporter gene can be treated with chemicals andbiologicals and those which have the capacity to stop the expression ofthe reporter gene can be identified.

The precursor cell in which Notch function has been activated issubjected to cell growth conditions to induce proliferation. Such cellgrowth conditions (e.g., cell culture medium, temperature, if growth isdone in vitro) can be any of those commonly known in the art.Preferably, both Notch activation and exposure to cell growth conditionsis carried out in vitro. Contacting the cell with a Notch functionagonist and exposing the cell to cell growth conditions can be carriedout concurrently or, if the agonist acts over a sufficient period oftime, sequentially (as long as Notch function activation to inhibitdifferentiation is present while cell growth occurs).

5.3.1. MODULATING OTHER SIGNALING PATHWAYS WITH NOTCH

Notch defines a general cell interaction mechanism whose biologicalfunction is to permit or block the action of developmental signals thatare essential for the progression of undifferentiated progenitor cellsto a more differentiated state. Consistent with that is the discoverythat one can modulate the activity of other signaling pathways bymodulating Notch. Thus, in another embodiment, the invention providesmethods of modulating other cell signal transduction pathway, e.g.,those that mediate cell growth and differentiation.

A dramatic example of how Notch signaling regulates specificdifferentiation pathways involves the Ras pathway in the developingDrosophila eye, which is used to transmit an inductive signal generatedby ligand-induced activation of the Sevenless receptor tyrosine kinase,and is blocked by appropriately timed activation of the Notch pathway.We have demonstrated that in the cone cell precursors of the developingDrosophila eye, Notch activation and Ras1-mediated signalling separatelycause opposite cell-fate alterations. Co-expression studies in thesecells demonstrate that Notch activation inhibits the neuraldifferentiation produced by constitutively activated components of awell-defined inductive signalling cascade, including the Sevenlessreceptor tyrosine kinase, Ras1 and Raf. Therefore, the activation ofNotch in a cell blocks the action of activated ras (see Section 6,infra).

Consistent with the notion that Notch activation initiates a distinctsignalling pathway that modulates the cellular response to signalstransduced by diverse pathways is the finding that the modulation ofNotch activity controls the action of Drosophila wingless (Hing et al.,1994, Mech. Dev. 47:261-268), a homologue of the mouse wnt-1 locus,which encodes a secreted protein involved in cell-signaling duringvarious stages in development (Nusslein-Volhard and Wieschaus, 1980,Nature 287:795-801; Martinez Arias et al., 1988, Development103:157-170; Nusse and Varmus, 1992, Cell 69:1073-1087; Struhl andBasler, 1993, Cell 72:527-540). Therefore, agonists and antagonists ofthe Notch pathway provide a novel and unique tool in manipulating theactivity of specific signals which control the differentiation of cellsusing pathways unrelated to Notch. In a particular embodiment of theinvention, cells are contacted with an agonist of Notch function toinhibit the function of a signaling pathway that regulates cell growthor differentiation.

5.4. NOTCH AND TERMINAL DIFFERENTIATION

The present invention is also directed to using agents to inhibit theNotch pathway such that cells, which are maintained in onedifferentiation state by Notch pathway activity, can be allowed tochange their differentiation state. Notch expression is generallyassociated with non-terminally differentiated cells. One exception tothis general rule is that Notch is expressed in post-mitotic neurons ofrat and human adult retina (Ahmad et al., unpublished results).Immunocytochemical staining data indicates that the Notch polypeptidesrecognized by the antibodies are nuclear. The expression of engineeredNotch fragments that are localized in the nuclear has been documented(reviewed in Artavanis-Tsakonas et al., 1995, Science 268:225-232), andthese fragments were shown to be associated with activated mutantphenotypes. The presence of an activated form of Notch in the nucleusmay lock these cells into a particular state of differentiation byrestricting or completely blocking their capacity to respond todifferentiation and/or proliferation stimuli. Therefore, it isconceivable that these post-mitotic neurons maintain theirdifferentiated state by virtue of an activated Notch-1 form that isindependent of Notch ligands. This state may perhaps afford such cellpopulations a certain plasticity. For example, an eventual cessation ofnuclear Notch-1 activity might allow these cells to re-enter a mitoticstate and/or respond to specific differentiation signals. In thiscontext, it is interesting to note that retinal neurons in lowervertebrates such as Goldfish and Xenopus have regenerative capacity.Chemical ablation of specific neurons, such as degeneration ofdopaminergic amacrine cells by 6-OH dopamine result in their replacementby regeneration (Reh and Tully, 1986, Dev. Biol. 114(2):463-469).However, such plasticity for regenerative purposes have not beenobserved in higher vertebrates. The observed Notch-1 activity in matureretinal neurons in the rat may represent the recapitulation of thefunctional significance of Notch-1 in retinal regeneration in lowervertebrates. The invention thus provides a method comprisingantagonizing Notch function to confer regenerative properties on themammalian neurons (e.g., of the central nervous system), thus leading toregeneration. Such a method comprises contacting a mammalian neuron withan antagonist of Notch function and exposing the neuron to neuronal cellgrowth conditions.

5.5. OBTAINING PRECURSOR CELLS

Precursor cells can be obtained by any method known in the art. Thecells can be obtained directly from tissues of an individual or fromcell lines or by production in vitro from less differentiated precursorcells, e.g., stem or progenitor cells. An example of obtaining precursorcells from less differentiated cells is described in Gilbert, 1991,Developmental Biology, 3rd Edition, Sinauer Associates, Inc., SunderlandMass. Briefly, progenitor cells can be incubated in the presence ofother tissues or growth and differentiation factors which cause the cellto differentiate. For example, when lung bud epithelium is culturedalone, no differentiation occurs. However, when lung bud epithelium iscultured with stomach mesenchyme or intestinal mesenchyme, the lung budepithelium differentiates into gastric glands or villi, respectively.Further, if lung bud epithelium is cultured with liver mesenchyme orbronchial mesenchyme, the epithelium differentiates into hepatic cordsor branching bronchial buds, respectively. Once a progenitor cell hasreached a desired differentiation state, a Notch function agonist can beused to stop differentiation.

5.5.1. ISOLATION OF STEM OR PROGENITOR CELLS

The following describes approaches which allow for the isolation ofprecursor cells and precursor cell-containing tissues, which are to betreated with agonists and, if subsequently desired, antagonists of theNotch pathway according to the present invention. As already alluded to,isolated cell types or even mixtures of cell populations can be treatedwith Notch function agonists. The isolated precursor cell or precursorcell population can be cultured ex vivo for proliferation which underthe influence of the Notch function agonists and cell growth conditionscan continue to divide, i.e., expand, in order to reach the desirednumbers before transplantation. Optionally, a recombinant gene can beintroduced into the cell so that it or its progeny expresses a desiredgene product before transplantation. Introduction of a recombinant genecan be accomplished either before or after precursor cell expansion.

In a preferred embodiment, the precursor cell populations are purifiedor at least highly enriched. However, in order to treat precursor cellswith Notch reagents it is not necessary that the precursor cells are apure population. Once a mixture is treated, only Notchpathway-expressing non-differentiated precursors will be refractory todifferentiation signals but will respond to growth signals while theirdifferentiated partners will eventually terminally differentiate andcease growing, such that the precursor cells will outgrow thedifferentiated cells and can be purified from the original mixedpopulation. Consequently, the precursor population can still be expandedselectively. Furthermore, purification may not be necessary or desirableprior to therapeutic administration in vivo.

The isolation of precursor cells for use in the present invention can becarried out by any of numerous methods commonly known to those skilledin the art. For example, one common method for isolating precursor cellsis to collect a population of cells from a patient and usingdifferential antibody binding, wherein cells of one or more certaindifferentiation stages are bound by antibodies to differentiationantigens, fluorescence activated cell sorting is used to separate thedesired precursor cells expressing selected differentiation antigensfrom the population of isolated cells. The following section describesexemplary methods for the isolation of various types of stem cells.

5.5.1.1. MESENCHYMAL STEM CELLS

One of the most important type of progenitor cells vis a vis fortherapeutic applications are those derived from the mesenchyme.Mesenchymal progenitors give rise to a very large number of distincttissues (Caplan, 1991, J. Orth. Res 641-650). Most work to date involvesthe isolation and culture of cells which can differentiate intochondrocytes and osteoblasts. The systems developed to isolate therelevant progenitor cell populations were worked out first in chickembryos (Caplan, 1970, Exp. Cell. Res. 62:341-355; Caplan, 1981, 39thAnnual Symposium of the Society for Developmental Biology, pp. 37-68;Caplan et al., 1980, Dilatation of the Uterine Cervix 79-98; DeLuca etal., 1977, J. Biol. Chem. 252:6600-6608; Osdoby et al., 1979, Dev. Biol.73:84-102; Syftestad et al., 1985, Dev. Biol. 110:275-283). Conditionswere defined under which chick mesenchymal cells differentiated intochondrocytes and bone. Id. With regard to cartilage and bone, theproperties of mouse or human mesenchymal limb appear to be quite similarif not identical (Caplan, 1991, J. Orth. Res. 641-650). Mesenchymalcells capable of differentiating into bone and cartilage have also beenisolated from marrow (Caplan, 1991, J. Orth. Res. 641-650).

Caplan et al., 1993, U.S. Pat. No. 5,226,914 describes an exemplarymethod for isolating mesenchymal stem cells from bone marrow. Theseisolated marrow stem cells can be used in conjunction with Notchreagents to expand the stem cell population. These expanded cells maythen be transplanted into a host where they can differentiate intoosteocytes, cartilage, chondocytes, adipocytes, etc., depending on thesurrounding microenvironment of the transplant site.

Animal models involving mice, rats as well as avian preparations, havesuggested that the source for mesenchymal stem cells is bone marrow. Ithas been possible to purify marrow mesenchymal cells by theirdifferential adhesion to culture dishes and demonstrate that they candifferentiate, e.g., into osteoblasts. Expansion of such isolated stemcells using Notch reagents can provide a source of cells which whentransplanted to the appropriate sites will be induced by themicroenvironment to differentiate into the appropriate lineage and helprepair damaged and/or diseased tissue. It is expected that the animalmodels described to date will be applicable to humans. Indeed, as far ascartilage and bone are concerned, the properties of mouse and human limbmesenchymal cells in culture are quite similar, if not identical(Hauska, 1974, Dev. Biol. 37:345-368; Owens and Solursh, 1981, Dev.Biol. 88:297-311). The isolation of human marrow and the demonstrationthat cells deriving from it can sustain osteogenesis has been described,e.g., by Bab et al., 1988, Bone Mineral 4:373-386.

Several bone marrow isolation protocols have been reported and can beused to obtain progenitor or precursor cells. Single cell suspensionsfrom rat bone marrow can be prepared according to Goshima et al., 1991,Clin. Orth. and Rel. Res. 262:298-311. Human stem cell cultures frommarrow can be prepared as described by Bab et al., 1988, Bone Mineral4:373-386 as follows: Whole marrow cells are obtained from fivepatients. The marrow samples are separated from either the iliac crestor femoral midshaft. Marrow samples, 3 ml in volume, are transferred to6 ml of serum-free Minimal Essential Medium (MEM) containing 50 U/mlpenicillin and 0.05 mg/ml streptomycin-sulfate. A suspension ofpredominantly single cells is prepared as described previously (Bab etal., 1984, Calcif. Tissue Int. 36:77-82; Ashton et al., 1984, Calcif.Tissue Int. 36:83-86) by drawing the preparation into a syringe andexpelling it several times sequentially through 19, 21, 23 and 25 gaugeneedles. The cells are counted using a fixed volume hemocytometer andthe concentration adjusted to 1-5×10⁸ total marrow cells per mlsuspension. Positive and negative control cell suspensions can be set asdescribed before (Shteyer et al., 1986, Calcif. Tissue Int. 39:49-54),using rabbit whole marrow and spleen cells, respectively.

5.5.1.2. NEURAL STEM CELLS

It is generally assumed that neurogenesis in the central nervous systemceases before or soon after birth. In recent years, several studies havepresented evidence indicating that at least to some degree new neuronscontinue to be added to the brain of adult vertebrates (Alvarez-Buyllaand Lois, 1995, Stem Cells (Dayt) 13:263-272). The precursors aregenerally located in the wall of the brain ventricles. It is thoughtthat from these proliferative regions, neuronal precursors migratetowards target positions where the microenvironment induces them todifferentiate. Studies have been reported where cells from thesub-ventricular zone can generate neurons both in vivo as well as invitro, reviewed in Alvarez-Buylla and Lois, 1995, Stem Cells (Dayt)13:263-272.

The neuronal precursors from the adult brain can be used as a source ofcells for neuronal transplantation (Alvarez-Buylla, 1993, Proc. Natl.Acad. Sci. USA 90:2074-2077). Neural crest cells have also been longrecognized to be pluripotent neuronal cells which can migrate anddifferentiate into different cell neuronal cell types according to theinstructions they receive from the microenvironment they find themselvesin (LeDouarin and Ziller, 1993, Curr. Opin. Cell Biol. 5:1036-1043).

5.5.1.3. FETAL CELLS

The fact that fetal brain tissue has been shown to have clear behavioraleffects when transplanted into adult lesioned brains, has focusedattention on human fetal tissue as a potential cell source intransplantation protocols designed to improve neurodegenerativedisorders (Bjorklund, 1993, Nature 362:414-415; McKay, 1991, TrendsNeurosci. 14:338-340). Nevertheless both ethical, as well as practicalconsiderations make fetal tissue a difficult source to deal with.Expansion of neuronal stem cells whether fetal or otherwise using Notchfunction agonists provides an alternative source for obtaining thedesired quantities of precursor cells for transplantation purposes.Fetal tissues or adult tissues containing precursors can be treated withNotch function agonists as described earlier in order to expand theundifferentiated progenitor cell populations. Fetal cells can placedinto primary culture using, for example, protocols developed by Sabateet al., 1995, Nature Gen. 9:256-260, before being treated with Notchfunction agonists. By way of example but not limitation, the procedureis as follows: Primary cultures of human fetal brain cells can beisolated from human fetuses, obtained from legal abortions after 5 to 12weeks of gestation. Expulsion can be done by syringe-driven gentleaspiration under echographic control. Fetuses collected in sterilehibernation medium are dissected in a sterile hood under astereomicroscope. Brains are first removed in toto in hibernation mediumcontaining penicillin G 500 U/ml, streptomycin 100 μg/ml, and fungizon 5μg/ml. For fetuses of six to eight weeks of age the brain is separatedinto an anterior (telencephalic vesicles and diencephalon) and aposterior fraction (mesencephalon, pons and cerebellar enlage) and aposterior in toto after careful removal of meninges. For older fetuses,striatal hippocampal, cortical and cerebellar zones expected to containproliferative precursor cells are visualized under the stereomicroscopeand dissected separately. Cells are transferred to either Opti-MEM(Gibco BRL) containing 15% heat-inactivated fetal bovine serum (FBS)(Seromed), or to a defined serum-free medium (DS-FM) with humanrecombinant bFGF (10 ng/ml, Boehringer), which is a minor modificationof the Bottenstein-Sato medium 39 with glucose, 6 g/l, glutamine 2 mM(Gibco BRL), insulin 25 ug/ml (Sigma) transferrin 100 μg/ml (Sigma),sodium selenite 30 nM (Gibco BRL), progesterone 20 nM (Sigma),putrescine 60 nM (Sigma), penicillin G (500 U/ml), streptomycin 100μg/ml, and fungizon 5 μg/ml. Cells, approximately 40,000 per cm², aregrown at 37° C. in an atmosphere containing 10% CO₂ in tissue culturedishes (Falcon or Nunc) coated with gelatin (0.25% wt/vol) followed byMatrigel (Gibco BRL, a basement membrane extract enriched in laminin andcontaining trace amounts of growth factors diluted one in 20). Cells inculture can be treated with Notch function agonists in order to expandthe population of the appropriate cells until the desired cell mass isreached for transplantation.

5.5.1.4. HEMATOPOIETIC STEM CELLS

Any technique which provides for the isolation, propagation, andmaintenance in vitro of hematopoietic stem cells (HSC) can be used inthis embodiment of the invention. Techniques by which this can beaccomplished include (a) the isolation and establishment of HSC culturesfrom bone marrow cells isolated from the future host, or a donor, or (b)the use of previously established long-term HSC cultures, which may beallogeneic or xenogeneic. Non-autologous HSC are used preferably inconjunction with a method of suppressing transplantation immunereactions of the future host/patient. In a particular embodiment of thepresent invention, human bone marrow cells can be obtained from theposterior iliac crest by needle aspiration (see, e.g., Kodo et al.,1984, J. Clin. Invest. 73:1377-1384). In a preferred embodiment of thepresent invention, the HSCs can be made highly enriched or insubstantially pure form. This enrichment can be accomplished before,during, or after long-term culturing, and can be done by any techniquesknown in the art. Long-term cultures of bone marrow cells can beestablished and maintained by using, for example, modified Dexter cellculture techniques (Dexter et al., 1977, J. Cell Physiol. 91:335) orWitlock-Witte culture techniques (Witlock and Witte, 1982, Proc. Natl.Acad. Sci. USA 79:3608-3612).

Another technique for the isolation of HSC is described by Milner etal., 1994, Blood 83:2057-2062. Bone marrow samples are obtained and areseparated by Ficoll-Hypaque density gradient centrifugation, are washed,and stained using two-color indirect immunofluorescent antibody bindingand then separated by fluorescence-activated cell sorting (FACS). Thecells are labelled simultaneously with IgG antibodies such that CD34⁺hematopoietic stem cells, including the immature subset that lacksexpression of individual lineage associated antigens, CD34⁺ lin⁻, areisolated from the cells collected from marrow.

Where hematopoietic progenitor cells are desired, the presence ofhematopoietic progenitor cells and/or their progeny can be detected bycommonly known in vitro colony forming assays (e.g., those that detectCFU-GM, BFU-E). As another example, assays for hematopoietic stem cellsare also known in the art (e.g., spleen focus forming assays, assaysthat detect the ability to form progenitors after replating).

5.5.1.5. EPITHELIAL STEM CELLS

Epithelial stem cells (ESCs) or keratinocytes can be obtained fromtissues such as the skin and the lining of the gut by known procedures(Rheinwald, 1980, Meth. Cell Bio. 21A:229). In stratified epithelialtissue such as the skin, renewal occurs by mitosis of precursor cellswithin the germinal layer, the layer closest to the basal lamina.Precursor cells within the lining of the gut provide for a rapid renewalrate of this tissue. ESCs or keratinocytes obtained from the skin orlining of the gut of a patient or donor can be grown in tissue culture(Rheinwald, 1980, Meth. Cell Bio. 21A:229; Pittelkow and Scott, 1986,Mayo Clinic Proc. 61:771). If the ESCs are provided by a donor, a methodfor suppression of host versus graft reactivity (e.g., irradiation, drugor antibody administration to promote moderate immunosuppression) canalso be used.

5.5.1.6. LIVER STEM CELLS

Liver stem cells can be isolated by methods described in PCT PublicationWO 94/08598, dated Apr. 28, 1994.

5.5.1.7. KIDNEY STEM CELLS

Mammalian kidney emerges from the metanephric mesenchyme which inducesthe uteric bud to undergo a series of morphogenetic movements ultimatelyforming the mature urinary collecting system (Nigam and Brenner, 1992,Curr. Opin. Nephrol. Huper 1:187-191. The uteric bud, an epithelialoutgrowth of the Wolfian duct, contracts and induces condensing adjacentmesenchyme along differentiation pathways of epithelial divergence inearly embryonic life. Attempts to study this process in vitro have beenreported; metanephros in organ culture can be induced to form tubulesusing embryonic spinal cord as the inducer. While the specifictransducing agents that lead to the induction of metanephric mesenchymeby the uteric bud in vivo or by spinal cord in vitro are not known, itis clear that differentiation program is induced in progenitor cells(Karp et al., 1994, Dev.

Biol. 91:5286-5290).

5.5.2. EXPANSION AND DIFFERENTIATION

After the precursors cells have been isolated according to the methodsdescribed above or other methods known in the art, the precursor cellscan be contacted with an amount of an agonist of Notch functioneffective to inhibit differentiation, and are exposed to cell growthconditions (e.g., promoting mitosis) such that the cell proliferates toobtain an expanded precursor population according to the presentinvention.

In one embodiment, substantially no differentiation of the precursorcells occurs during expansion. The amount of differentiation that occurscan be assayed for by known assays, e.g., those that detect the presenceof more differentiated cells by detecting functions associated with aparticular stage of differentiation, e.g., expression of differentiationantigens on the cell surface or secretion of proteins associated with aparticular state, or ability to generate various cell types, ordetecting morphology associated with particular stages ofdifferentiation.

Once the population has reached a desired titer, the Notch functionagonist can be removed (e.g., by separation, dilution), or a Notchfunction antagonist can be added, such that Notch function is absent orinhibited allowing at least some of the cells in the expanded populationto differentiate in the presence of the desired differentiation signalsto a desired differentiation state or to a differentiation state of thecell such that the cell expresses a desired phenotype. Optionally, oncethe cells reach the desired differentiation state the Notch pathway canagain be activated with a Notch agonist to freeze the cell in thatdifferentiation state. The cells can be differentiated to a terminallydifferentiated state if the function of that terminally differentiatedcell is desired.

5.6. GENE THERAPY

The cells produced by manipulation of the Notch pathway can be maderecombinant and used in gene therapy. In its broadest sense, genetherapy refers to therapy performed by the administration of a nucleicacid to a subject. The nucleic acid, either directly or indirectly viaits encoded protein, mediates a therapeutic effect in the subject. Thepresent invention provides methods of gene therapy wherein a nucleicacid encoding a protein of therapeutic value (preferably to humans) isintroduced into the precursor cells expanded according to the invention,before or after expansion, such that the nucleic acid is expressible bythe precursor cells and/or their progeny, followed by administration ofthe recombinant cells to a subject.

The recombinant precursor cells of the present invention can be used inany of the methods for gene therapy available in the art. Thus, thenucleic acid introduced into the cells may encode any desired protein,e.g., a protein missing or dysfunctional in a disease or disorder. Thedescriptions below are meant to be illustrative of such methods. It willbe readily understood by those of skill in the art that the methodsillustrated represent only a sample of all available methods of genetherapy.

For general reviews of the methods of gene therapy, see Goldspiel etal., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596;Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann.Rev. Biochem. 62:191-217; May, 1993, TIBTECH 11(5):155-215). Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), 1993, Current Protocols inMolecular Biology, John Wiley & Sons, NY; and Kriegler, 1990, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY.

In an embodiment in which recombinant precursor cells are used in genetherapy, a gene whose expression is desired in a patient is introducedinto the precursor cells such that it is expressible by the cells and/ortheir progeny, and the recombinant cells are then administered in vivofor therapeutic effect.

Precursor cells or expanded precursor cells can be used in anyappropriate method of gene therapy, as would be recognized by those inthe art upon considering this disclosure. The resulting action of arecombinant precursor cell or its progeny cells administered to apatient can, for example, lead to the activation or inhibition of apre-selected gene in the patient, thus leading to improvement of thediseased condition afflicting the patient.

The desired gene is transferred to precursor cells in tissue culture bysuch methods as electroporation, lipofection, calcium phosphate mediatedtransfection, or viral infection. Usually, the method of transferincludes the transfer of a selectable marker to the cells. The cells arethen placed under selection to isolate those cells that have taken upand are expressing the transferred gene. Those precursor cells are thendelivered to a patient.

In this embodiment, the desired gene is introduced into a precursor cellprior to administration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the gene sequences, cell fusion, chromosome-mediated genetransfer, microcell-mediated gene transfer, spheroplast fusion, etc.Numerous techniques are known in the art for the introduction of foreigngenes into cells (see e.g., Loeffler and Behr, 1993, Meth. Enzymol.217:599-618; Cohen et al., 1993, Meth. Enzymol. 217:618-644; Cline,1985, Pharmac. Ther. 29:69-92) and may be used in accordance with thepresent invention, provided that the necessary developmental andphysiological functions of the recipient cells are not disrupted. Thetechnique should provide for the stable transfer of the gene to thecell, so that the gene is expressible by the cell and preferablyheritable and expressible by its cell progeny.

One common method of practicing gene therapy is by making use ofretroviral vectors (see Miller et al., 1993, Meth. Enzymol.217:581-599). A retroviral vector is a retrovirus that has been modifiedto incorporate a preselected gene in order to effect the expression ofthat gene. It has been found that many of the naturally occurring DNAsequences of retroviruses are dispensable in retroviral vectors. Only asmall subset of the naturally occurring DNA sequences of retroviruses isnecessary. In general, a retroviral vector must contain all of thecis-acting sequences necessary for the packaging and integration of theviral genome. These cis-acting sequences are:

a) a long terminal repeat (LTR), or portions thereof, at each end of thevector;

b) primer binding sites for negative and positive strand DNA synthesis;and

c) a packaging signal, necessary for the incorporation of genomic RNAinto virions.

The gene to be used in gene therapy is cloned into the vector, whichfacilitates delivery of the gene into a precursor cell by infection ordelivery of the vector into the cell.

More detail about retroviral vectors can be found in Boesen et al.,1994, Biotherapy 6:291-302, which describes the use of a retroviralvector to deliver the mdrl gene to hematopoietic stem cells in order tomake the stem cells more resistant to chemotherapy. Other referencesillustrating the use of retroviral vectors in gene therapy are: Cloweset al., 1994, J. Clin. Invest. 93:644-651; Kiem et al., 1994, Blood83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141;and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel.3:110-114.

Adenoviruses are also of use in gene therapy. Adenoviruses areespecially attractive vehicles for delivering genes to respiratoryprecursor cells. Adenoviruses can also be used to deliver genes toprecursor cells from the liver, the central nervous system, endothelium,and muscle. Adenoviruses have the advantage of being capable ofinfecting non-dividing cells. Kozarsky and Wilson, 1993, Current Opinionin Genetics and Development 3:499-503 present a review ofadenovirus-based gene therapy. Other instances of the use ofadenoviruses in gene therapy can be found in Rosenfeld et al., 1991,Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155; andMastrangeli et al., 1993, J. Clin. Invest. 91:225-234.

It has been proposed that adeno-associated virus (AAV) be used in genetherapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300).

A desired gene can be introduced intracellularly and incorporated withinhost precursor cell DNA for expression, by homologous recombination(Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935;Zijlstra et al., 1989, Nature 342:435-438).

In a specific embodiment, the desired gene recombinantly expressed inthe precursor cell to be introduced for purposes of gene therapycomprises an inducible promoter operably linked to the coding region,such that expression of the recombinant gene is controllable bycontrolling the presence or absence of the appropriate inducer oftranscription.

In another embodiment, if a greater number of differentiated cells isdesired before administering to a patient then the precursor cells canbe differentiated prior to expansion. In another embodiment, one canexpand and differentiate the precursor cells simultaneously such thatgreater numbers of differentiated cells are obtained.

5.7. PHARMACEUTICAL COMPOSITIONS

The invention provides methods of treatment by administration to asubject of a pharmaceutical (therapeutic) composition comprising atherapeutically effective amount of a recombinant or non-recombinantcell, preferably a stem or progenitor cell. Such a stem cell orrecombinant stem cell envisioned for therapeutic use is referred tohereinafter as a "Therapeutic" or "Therapeutic of the invention." In apreferred aspect, the Therapeutic is substantially purified. The subjectis preferably an animal, including but not limited to animals such ascows, pigs, horses, chickens, cats, dogs, etc., and is preferably amammal, and most preferably human.

The present invention provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of aTherapeutic, and a pharmaceutically acceptable carrier or excipient.Such a carrier includes but is not limited to saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof. Thecarrier and composition can be sterile. The formulation should suit themode of administration.

The composition, if desired, can also contain minor amounts of wettingor emulsifying agents, or pH buffering agents. The composition can be aliquid solution, suspension, or emulsion.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection.

5.8. TRANSPLANTATION

The expanded stem cell populations of the present invention whetherrecombinantly expressing a desired gene or not can be transplanted intoa patient for the treatment of disease or injury or for gene therapy byany method known in the art which is appropriate for the type of stemcells being transplanted and the transplant site. Hematopoietic stemcells can be transplanted intravenously, as can liver stem cells whichwill locate to the liver. Neural stem cells can be transplanted directlyinto the brain at the site of injury or disease.

Methods of introduction include but are not limited to intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,and epidural routes. The compounds may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local. Inaddition, it may be desirable to introduce the pharmaceuticalcompositions of the invention into the central nervous system by anysuitable route, including intraventricular and intrathecal injection;intraventricular injection may be facilitated by an intraventricularcatheter, for example, attached to a reservoir, such as an Ommayareservoir.

In a specific embodiment, it may be desirable to administer theTherapeutics of the invention locally to the area in need of treatment;this may be achieved by, for example, and not by way of limitation,local infusion during surgery, topical application, e.g., in conjunctionwith a wound dressing after surgery, by injection, by means of acatheter, or by means of an implant, said implant being of a porous,non-porous, or gelatinous material, including membranes, such assialastic membranes, or fibers.

The following describes exemplary methods which can be modified for thetransplantation of precursor cells: Protocols for the isolation andtransplantation of fetal tissues in humans have been reported andclinical trials involving these studies having been carried out. Forexample, Lindvall et al., 1990, Science 247:574-577, have describedresults regarding grafts and survival of fetal dopamine neurons aftertransplantation into brain. Rinsing and partial dissociation ofprecursor cells, if necessary, can be carried out by a modification ofthat described in Lindvall et al., 1989, Arch. Neurol. 46:615.

By way of example, implantation of cells into the brain can be performedas follows. Implantation is done at three sites in the left putamen witha stereotactic technique (Lindvall et al., 1989, Arch. Neurol. 46:615).For each site, 20 μl of the dissociated cells is drawn into theinstrument (outer diameter, 1.0 mm). The cells are injected along a 10,12 and 14 mm linear tract, respectively, in either 2.5 μl portions for15 to 20 seconds each. Between each injection there is a 2 minute delay,and the cannula is then retracted 1.5 to 1.7 mm. After the finalinjection, the cannula is left in situ for 8 minutes before being slowlywithdrawn from the brain. After surgery the cell viability is assessedfollowing the procedure of Brundin et al., 1985, Brain. Res. 331:251.

Another example is outlined by Caplan et al., 1993, U.S. Pat. No.5,226,914. Briefly, after marrow cells are harvested from bone marrowplugs and the marrow mesenchymal, stem cells are separated bycentrifugation. The stem cells are isolated further by selectiveadherence to the plastic or glass surface of a tissue culture dish. Thestem cells are allowed to proliferate but not differentiate. Porousceramic cubes composed of 60% hydroxyapatite and 40% β-tricalciumphosphate are added to the cells under a slight vacuum. The cubes withadhered cells are implanted into incisional pockets along the backs ofnude mice. The mesenchymal stem cells differentiate into bone.

The titer of stem cells transplanted or the amount of the Therapeutic ofthe invention which will be effective in the treatment of a particulardisorder or condition will depend on the nature of the disorder orcondition, and can be determined by standard clinical techniques. Inaddition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.

6. INHIBITION OF RAS-1-MEDIATED SIGNALING BY ACTIVATED NOTCH INDROSOPHILA EYE

In the cone cell precursors of the developing Drosophila eye, Notchactivation and Ras1-mediated signaling separately cause oppositecell-fate alterations. Co-expression studies in these cells demonstratethat Notch activation inhibits the neural differentiation produced byconstitutively activated components of a well-defined inductivesignaling cascade, including the Sevenless receptor tyrosine kinase,Ras1 and Raf.

The sevenless signaling pathway is required only for the induction ofthe R7 photoreceptor cell by the previously determined R8 cell of eachommatidium. The interaction of the sevenless gene product and itsligand, encoded by the bride of sevenless (boss) gene, normally occursonly between two particular cell types during a narrow developmentaltime window (Basler and Hafen, 1989, Development 107:723-731; Mullinsand Rubin, 1991, Proc. Natl. Acad. Sci. USA 88:9387-9391; Kramer et al.,1991, Nature 352:207-212). Flies mutant for either or both genes displaya very specific phenotype: misrouting of the R7 cell precursor into thecone-cell fate (Tomlinson and Ready, 1986, Science 231:400-402, 1987,Dev. Biol. 123:264-275; Reinke and Zipursky, 1988, Cell 55:321-330).Within each ommatidium, sevenless is expressed in a small set of cellsseparated by no more than a few cell diameters, consisting of the R3,R4, and R7 precursor cells, the four cone cell precursor cells, and upto two so-called `mystery cells` (Tomlinson et al., 1987, Cell51:143-150; Bowtell et al., 1989, Proc. Natl. Acad. Sci. USA86:6245-6249; Basler et al., 1989, EMBO J. 8:2381-2386). In wild-typeflies, only the R7 precursor cell ever comes into contact with the R8cell, which is the only eye disc cell type that expresses bride ofsevenless, resulting in the recruitment of one R7 cell per ommatidium(Kramer et al., 1991, Nature 352:207-212). Experiments in which sev andboss were expressed ubiquitously under heat-shock gene control havedemonstrated that the spatially restricted presentation of ligand by theR8 cell is a crucial feature of this inductive signalling mechanism(Basler and Hafen, 1989, Science 243:931-934; Bowtell et al., 1989, Cell56:931-936; Van Vactor et al., 1991, Cell 67:1145-1155). Recent studieshave shown that activation of the Sevenless receptor tyrosine kinaseinitiates a signaling cascade involving the activation of Ras1 and thesubsequent activation of Raf (Simon et al., 1991, Cell 67:701-716;Bonfini et al., 1992, Science 255:603-606; Dickson et al., 1992, GenesDev. 6:2327-2339; Dickson et al., Nature 360:600-603). Ras1 and Raf arealso downstream targets of other receptor tyrosine kinases inDrosophila, including the torso kinase and the Drosophila EGF receptorhomolog (Ambrosio et al., 1989, Nature 342:288-291; Simon et al., 1991,Cell 67:701-716; Doyle and Bishop, 1993, Genes Dev. 7:633-646; Melnicket al., 1993, Development 118:127-138; Diaz-Benjumea and Hafen, 1994,Development 120:569-578).

In contrast to sevenless-mediated signalling, the signalling mechanisminvolving Notch appears to regulate a common step in cell-fatecommitment throughout development. The Drosophila Notch gene encodes alarge transmembrane receptor protein with an extracellular domainconsisting of 36 tandem EGF-like repeats and 3 Notch/lin-12 repeats aswell as an intracellular domain containing 6 tandem cdc10/ankyrinrepeats (Wharton et al., 1985, Cell 43:567-581; Kidd et al., 1986, Mol.Cell. Biol. 6:3094-3108). Unlike the sev and boss gene products, theDrosophila Notch protein is widely expressed in developing tissues,including all or most cells of the imaginal eye disc (Johansen et al.,1989, J. Cell Biol. 109:2427-2440; Kidd et al., 1989, Genes Dev.3:1113-1129; Fehon et al., 1991, J. Cell. Biol. 113:657-669). Analysisof Notch gene mutant phenotypes has revealed that Notch function isrequired for numerous developmental processes, including embryonicneurogenesis (Poulson, 1937, Proc. Natl. Acad. Sci. USA 23:133-137,1940, J. Exp. Zool. 83:271-325), mesoderm differentiation (Corbin etal., 1991, Cell 67:311-323), axonal pathfinding (Giniger et al., 1993,Development 117:431-440), oogenesis (Ruohola et al., 1991, Cell66:433-449; Xu et al., 1992, Development 115:913-922; Cummings andCronmiller, 1994, Development 120:381-394), and differentiation of adultperipheral nervous system structures (Cagan and Ready, 1989, Genes Dev.3:1099-1112; Palka et al., 1990, Development 109:167-175; Hartensteinand Posakony, 1990, Dev. Biol. 142:13-30; Hartenstein et al., 1992,Development 116:1203-1220). A detailed study of the phenotypic effectsof the conditional loss-of-function allele Notch^(ts1) has shown thatevery cell type of the adult eye, including the R7 cell, requires Notchactivity at some stage for its proper cell-fate specification (Cagan andReady, 1989, Genes Dev. 3:1099-1112).

In the absence of boss gene function, the sevenless-expressing cells ofeach ommatidium may be induced to differentiate as neurons by ectopicactivation of the Sevenless protein, Ras1, or Raf (Basler et al., 1991,Cell 64:1069-1081; Fortini et al., 1992, Nature 355:559-561; Dickson etal., 1992, Genes Dev. 6:2327-2339; Dickson et al., Nature 360:600-603).Evidence is presented below that the neural induction of these cells byactivated sevenless pathway components is blocked by constitutive Notchactivation in the developing eye imaginal disc. These results indicatethat the signal mediated by Notch and its ligands are integrated withthe cell type-specific inductive signal mediated by Sevenless at a pointdownstream of Raf during R7 photoreceptor cell fate specification. Sinceboth Ras1 and Raf are utilized by other tissue-specific inductivesignalling pathways, our data implies that Notch may exert regulatoryeffects on these pathways as well.

6.1. MATERIALS AND METHODS

Drosophila culture

Flies were grown on standard medium at 18° C. for optimal imaginal discgrowth.

Immunohistochemistry

Antibody staining of eye imaginal discs was performed as described inGaul et al., 1992, Cell 68:1007-1019. For the Notch/ELAV stainings,mouse mAb C17.9C6 (Fehon et al., 1990, Cell 61:523-534) and rat mAb7E8A10 (Robinow and White, 1991, J. Neurobiol. 22:443-461) were used at1:2000 and 1:1 dilutions, respectively. For the Notch/Sevenless doublestainings, rat polyclonal Ab Rat5 (R. G. Fehon, I. Rebay, and S.Artavanis-Tsakonas, unpublished) and mouse mAb sev150C3 (Banerjee etal., 1987, Cell 51:151-158) were used at 1:500 and 1:1000 dilutions,respectively. In both cases, goat anti-mouse FITC-conjugated and goatanti-rat Texas Red-conjugated double-label grade secondary antibodies(Jackson ImmunoResearch Laboratories, Inc.) were used at 1:250 and 1:500dilutions, respectively.

Confocal microscopy

Confocal microscopy and image processing were performed as described byXu et al., 1992, Development 115:913-922.

6.2. RESULTS

Previous studies on R7 photoreceptor cell determination in Drosophilahave shown that specification of neural fate in the R7 precursor cell isinitiated by ligand-induced activation of the receptor tyrosine kinaseencoded by sevenless (reviewed in Greenwald and Rubin, 1992, Cell68:271-281), which is expressed strongly in a subset of uncommittedcells in each developing ommatidium, namely the R3, R4, and R7 precursorcells, the four cone cell precursors, and up to two so-called `mysterycells` (Tomlinson et al., 1987, Cell 51:143-150; Bowtell et al., 1989,Proc. Natl. Acad. Sci. USA 86:6245-6249; Basler et al., 1989, EMBO J.8:2381-2386). Activation of the Sevenless tyrosine kinase results in thesubsequent activation of Ras1 (Simon et al., 1991, Cell 67:701-716;Bonfini et al., 1992, Science 255:603-606), which in turn activates Raf(Dickson et al., 1992, Nature 360:600-603). These studies have also ledto the production of transgenic fly lines bearing constitutivelyactivated Sevenless, Ras1, and Raf proteins, all expressed undersevenless gene control in the above mentioned cells (Basler et al.,1991, Cell 64:1069-1081; Fortini et al., 1992, Nature 355:559-561;Dickson et al., 1992, Genes Dev. 6:2327-2339; Dickson et al., Nature360:600-603). In each case, expression of the activated sevenlesspathway component drives sevenless-expressing cells into neural fates,as judged by the expression of neural-specific antigens such as BP-104(Hortsch et al., 1990, Neuron 4:697-709) or ELAV (Bier et al., 1988,Science 240:913-916; Robinow and White, 1991, J. Neurobiol. 22:443-461)in the eye disc. While the wild-type Notch gene is expressed in andrequired for normal development of all or most eye disc cells (Cagan andReady, 1989, Genes Dev. 3:1099-1112; Fehon et al., 1991, J. Cell. Biol.113:657-669), a constitutively activated Notch receptor lacking theextracellular and transmembrane domains expressed under sevenless genecontrol blocks cell-fate commitment, preventing ELAV expression inneural precursors and causing cell-fate misspecifications among thesevenless-expressing cells (Fortini et al., 1993, Nature 365:555-557).

To determine whether the block imposed by activated Notch upon neuraldifferentiation can be circumvented by constitutive activation of any ofthe sevenless signalling pathway components, transgenic flies wereproduced co-expressing activated Notch and activated sevenless pathwayfactors in the sevenless-expressing cells. Eye discs of these flies weredouble-stained with antibodies directed against Notch and against theELAV protein to determine whether cells expressing activated Notch arecapable of neural induction by activated Sevenless, activated Ras, oractivated Raf. Since ELAV is a nuclear antigen, we chose to use anactivated Notch construct, termed sev-Notch^(nucl), that producesnuclear Notch protein localization (Fortini et al., 1993, Nature365:555-557). This nuclear Notch expression is easily distinguished fromthe apical membrane distribution of the endogenous wild-type Notchprotein (Fehon et al., 1991, J. Cell. Biol. 113:657-669; Fortini et al.,1993, Nature 365:555-557). Nuclear translocation of the Notch proteinapparently is not required for its activated behavior, since the samephenotypic effects are caused by a truncated Notch protein lackingextracellular but not transmembrane sequences that is apicallylocalized, as judged by antibody staining experiments (Fortini et al.,1993, Nature 365:555-557).

The analysis was restricted to the four cone cell precursors of eachdeveloping ommatidium for the following reasons. First, the cone cellprecursor nuclei are easily identified by their distinctivesausage-shaped morphology. Second, the cone cell precursors are normallynon-neural and thus should only be ELAV-positive as a result of thetransgene-driven activated sevenless pathway components. Third, Notchexpression in sev-Notch^(nucl) cone cell precursor nuclei persiststhroughout those ommatidial rows of the posterior eye disc in which conecell precursors exhibit strong ELAV expression if they are transformedinto neurons (Fortini et al., 1992, Nature 355:559-561, 1993, Nature365:555-557; Gaul et al., 1992, Cell 68:1007-1019; Dickson et al., 1992,Nature 360:600-603). By contrast, the nuclear Notch expression in R3 andR4 precursor cells and mystery cells is more transient, subsiding priorto the onset of ELAV expression (Fortini et al., 1993, Nature365:555-557).

Notch-positive cone cell precursor nuclei were scored for ELAVexpression in sev-Notchnucl flies also carrying either the activatedSevenless tyrosine kinase construct sev-S11 (Basler et al., 1991, Cell64:1069-1081), the activated Ras1 construct sevRas1^(Va112) (Fortini etal., 1992, Nature 355:559-561), or the activated Raf constructsE-raf^(torY9) (Dickson et al., 1992, Genes Dev. 6:2327-2339; Dickson etal., Nature 360:600-603). For each genotype, 500 Notch-positive conecell precursor nuclei in ommatidial rows 15-25 were examined,representing at least six separate pairs of eye discs. In no case did weobserve any cone cell precursor nuclei positive for both Notch and ELAVantigens (FIGS. 5A-5F). We frequently found that not all four cone cellprecursor nuclei in an ommatidium express Notch, and that those which donot are often ELAV-positive (FIGS. 5A-5F). These nuclei presumablycorrespond to cone cell precursors that do not express sev-Notch^(nucl)efficiently but do express sufficient amounts of an activated sevenlesspathway molecule to induce neural differentiation. Identical resultswere obtained with different sev-Notch^(nucl), sev-S11, andsevRas1^(Va112) transgenic lines as well as with an alternativeactivated Raf construct sE-raf^(tor4021) (Dickson et al., 1992, GenesDev. 6:2327-2339; Dickson et al., Nature 360:600-603).

To rule out the possibility that our failure to detect co-expression ofactivated Notch and ELAV antigens in cone cell precursor nuclei is dueto some mechanism that prevents two different sevenless promoterconstructs from being expressed in the same cell, we double-stained eyediscs of transgenic flies bearing both sev-Notchnucl and sev-S11 in asevenless^(d2) genetic background with antibodies against Notch andagainst the intracellular 60-kD subunit of Sevenless (Banerjee et al.,1987, Cell 51:151-158). Since sevenless^(d2) flies do not express thissubunit (Banerjee et al, 1987), the only Sevenless immunoreactivitydetected corresponds to the extracellularly truncated protein producedby the sev-S11 transgene (Basler et al., 1991, Cell 64:1069-1081). Itwas found that most cone cell precursors showing strong nuclearexpression of activated Notch also display strong apical membraneexpression of activated Sevenless, demonstrating that both transgenesare coexpressed in these cells (FIGS. 6A-6B).

6.3. DISCUSSION

The class of transmembrane receptor proteins encoded by the Notch locusand related genes appears to regulate a common step in cell-fateselection in organisms ranging from nematodes to humans (reviewed inGreenwald and Rubin, 1992, Cell 68:271-281; Fortini andArtavanis-Tsakonas, 1993, Cell 75:1245-1247). In many different celltypes, the signal generated by Notch activation renders cellstemporarily unable to respond to developmental cues from neighboringcells (Coffman et al., 1993, Cell 73:659-671; Rebay et al., 1993, Cell74:319-329; Struhl et al., 1993, Cell 74:331-345; Fortini et al., 1993,Nature 365:555-557; Lieber et al., 1993, Genes Dev. 7:1949-1965). TheNotch protein and its ligands Delta and Serrate may thus be part of ageneral mechanism that limits the competence of undifferentiated cellsto undergo cell-fate commitment (Coffman et al., 1993, Cell 73:659-671;Fortini and Artavanis-Tsakonas, 1993, Cell 75:1245-1247). This mechanismmay play a crucial role in the timing of inductive events by allowing anuncommitted cell to ignore irrelevant signals from adjacent cells untilit is presented with the appropriate inductive signal, presumablypreceded or accompanied by a signal that inactivates Notch in therecipient cell. Consistent with this notion, genetic analyses inCaenorhabditis and Drosophila have revealed an interdependence betweenNotch-mediated signaling and several distinct cell type-specificinductive signaling events (reviewed in Horvitz and Sternberg, 1991,Nature 351:535-541; Artavanis-Tsakonas and Simpson, 1991, Trends Genet.7:403-408; Greenwald and Rubin, 1992, Cell 68:271-281), although littleis known about how the different signals are integrated at the molecularlevel. We have sought to address this question by performing epistasistests between a constitutively activated Notch receptor and variousactivated components of the inductive signalling pathway involving theSevenless receptor tyrosine kinase, Ras1 and Raf in the developingDrosophila eye.

The results presented here indicate that the Notch receptor protein, inits active state, interferes with the intracellular signal generated byconstitutively activated versions of Sevenless, Ras1, and Raf (FIG. 7).Our epistasis data are difficult to reconcile with models in which theNotch protein mediates cell signaling primarily by promoting celladhesion (Hoppe and Greenspan, 1986, Cell 46:773-783; Greenspan, 1990,New Biologist 2:595-600) or by recruiting cell type-specific receptorsand their ligands to specialized membrane regions of polarized epithelia(Singer, 1992, Science 255:1671-1677). Instead, Notch apparentlymediates a separate signalling pathway whose input is integrated withthat of the Ras1 pathway at some point downstream of Raf, at least inthis case. Ras1 and Raf, unlike the sevenless receptor tyrosine kinase,act in many different tissues throughout Drosophila development, as doesNotch. For example, genetic studies have identified both Ras1 and Raf asessential components of the signaling pathways initiated by the torsoand Drosophila EGF receptor (DER) tyrosine kinases (Ambrosio et al.,1989, Nature 342:288-291; Simon et al., 1991, Cell 67:701-716; Doyle andBishop, 1993, Genes Dev. 7:633-646; Melnick et al., 1993, Development118:127-138; Diaz-Benjumea and Hafen, 1994, Development 120:569-578).Moreover, cell-fate specifications involving other types of signallingmolecules, such as the Drosophila scabrous, wingless and daughterlessgene products, also depend upon Notch gene function (Baker et al., 1990,Science 250:1370-1377; Hing et al., 1994, Mech. Dev., in press; Cummingsand Cronmiller, 1994, Development 120:381-394). Thus, the activity stateof Notch is likely to play an important regulatory role in modulatingsignalling by Ras1, Raf, and other signalling molecules in a variety ofdevelopmental processes.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 4                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1015 amino acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: hum N (Human Notch 2)                                           (B) LOCATION: 1155...2169                                                     (D) OTHER INFORMATION: Highly conserved ankyrin repeat                        region of Notch                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       SerAsnProCysGlnHisGlyAlaThrCysSerAspPheIleGlyGly                              151015                                                                        TyrArgCysGluCysValProGlyTyrGlnGlyValAsnCysGluTyr                              202530                                                                        GluValAspGluCysGlnAsnGlnProCysGlnAsnGlyGlyThrCys                              354045                                                                        IleAspLeuValAsnHisPheLysCysSerCysProProGlyThrArg                              505560                                                                        GlyLeuLeuCysGluGluAsnIleAspAspCysAlaArgGlyProHis                              65707580                                                                      CysLeuAsnGlyGlyGlnCysMetAspArgIleGlyGlyTyrSerCys                              859095                                                                        ArgCysLeuProGlyPheAlaGlyGluArgCysGluGlyAspIleAsn                              100105110                                                                     GluCysLeuSerAsnProCysSerSerGluGlySerLeuAspCysIle                              115120125                                                                     GlnLeuThrAsnAspTyrLeuCysValCysArgSerAlaPheThrGly                              130135140                                                                     ArgHisCysGluThrPheValAspValCysProGlnMetProCysLeu                              145150155160                                                                  AsnGlyGlyThrCysAlaValAlaSerAsnMetProAspGlyPheIle                              165170175                                                                     CysArgCysProProGlyPheSerGlyAlaArgCysGlnSerSerCys                              180185190                                                                     GlyGlnValLysCysArgLysGlyGluGlnCysValHisThrAlaSer                              195200205                                                                     GlyProArgCysPheCysProSerProArgAspCysGluSerGlyCys                              210215220                                                                     AlaSerSerProCysGlnHisGlyGlySerCysHisProGlnArgGln                              225230235240                                                                  ProProTyrTyrSerCysGlnCysAlaProProPheSerGlySerArg                              245250255                                                                     CysGluLeuTyrThrAlaProProSerThrProProAlaThrCysLeu                              260265270                                                                     SerGlnTyrCysAlaAspLysAlaArgAspGlyValCysAspGluAla                              275280285                                                                     CysAsnSerHisAlaCysGlnTrpAspGlyGlyAspCysSerLeuThr                              290295300                                                                     MetGluAsnProTrpAlaAsnCysSerSerProLeuProCysTrpAsp                              305310315320                                                                  TyrIleAsnAsnGlnCysAspGluLeuCysAsnThrValGluCysLeu                              325330335                                                                     PheAspAsnPheGluCysGlnGlyAsnSerLysThrCysLysTyrAsp                              340345350                                                                     LysTyrCysAlaAspHisPheLysAspAsnHisCysAsnGlnGlyCys                              355360365                                                                     AsnSerGluGluCysGlyTrpAspGlyLeuAspCysAlaAlaAspGln                              370375380                                                                     ProGluAsnLeuAlaGluGlyThrLeuValIleValValLeuMetPro                              385390395400                                                                  ProGluGlnLeuLeuGlnAspAlaArgSerPheLeuArgAlaLeuGly                              405410415                                                                     ThrLeuLeuHisThrAsnLeuArgIleLysArgAspSerGlnGlyGlu                              420425430                                                                     LeuMetValTyrProTyrTyrGlyGluLysSerAlaAlaMetLysLys                              435440445                                                                     GlnArgMetThrArgArgSerLeuProGlyGluGlnGluGlnGluVal                              450455460                                                                     AlaGlySerLysValPheLeuGluIleAspAsnArgGlnCysValGln                              465470475480                                                                  AspSerAspHisCysPheLysAsnThrAspAlaAlaAlaAlaLeuLeu                              485490495                                                                     AlaSerHisAlaIleGlnGlyThrLeuSerTyrProLeuValSerVal                              500505510                                                                     ValSerGluSerLeuThrProGluArgThrGlnLeuLeuTyrLeuLeu                              515520525                                                                     AlaValAlaValValIleIleLeuPheIleIleLeuLeuGlyValIle                              530535540                                                                     MetAlaLysArgLysArgLysHisGlySerLeuTrpLeuProGluGly                              545550555560                                                                  PheThrLeuArgArgAspAlaSerAsnHisLysArgArgGluProVal                              565570575                                                                     GlyGlnAspAlaValGlyLeuLysAsnLeuSerValGlnValSerGlu                              580585590                                                                     AlaAsnLeuIleGlyThrGlyThrSerGluHisTrpValAspAspGlu                              595600605                                                                     GlyProGlnProLysLysValLysAlaGluAspGluAlaLeuLeuSer                              610615620                                                                     GluGluAspAspProIleAspArgArgProTrpThrGlnGlnHisLeu                              625630635640                                                                  GluAlaAlaAspIleArgArgThrProSerLeuAlaLeuThrProPro                              645650655                                                                     GlnAlaGluGlnGluValAspValLeuAspValAsnValArgGlyPro                              660665670                                                                     AspGlyCysThrProLeuMetLeuAlaSerLeuArgGlyGlySerSer                              675680685                                                                     AspLeuSerAspGluAspGluAspAlaGluAspSerSerAlaAsnIle                              690695700                                                                     IleThrAspLeuValTyrGlnGlyAlaSerLeuGlnAlaGlnThrAsp                              705710715720                                                                  ArgThrGlyGluMetAlaLeuHisLeuAlaAlaArgTyrSerArgAla                              725730735                                                                     AspAlaAlaLysArgLeuLeuAspAlaGlyAlaAspAlaAsnAlaGln                              740745750                                                                     AspAsnMetGlyArgCysProLeuHisAlaAlaValAlaAlaAspAla                              755760765                                                                     GlnGlyValPheGlnIleLeuIleArgAsnArgValThrAspLeuAsp                              770775780                                                                     AlaArgMetAsnAspGlyThrThrProLeuIleLeuAlaAlaArgLeu                              785790795800                                                                  AlaValGluGlyMetValAlaGluLeuIleAsnCysGlnAlaAspVal                              805810815                                                                     AsnAlaValAspAspHisGlyLysSerAlaLeuHisTrpAlaAlaAla                              820825830                                                                     ValAsnAsnValGluAlaThrLeuLeuLeuLeuLysAsnGlyAlaAsn                              835840845                                                                     ArgAspMetGlnAspAsnLysGluGluThrProLeuPheLeuAlaAla                              850855860                                                                     ArgGluGlySerTyrGluAlaAlaLysIleLeuLeuAspHisPheAla                              865870875880                                                                  AsnArgAspIleThrAspHisMetAspArgLeuProArgAspValAla                              885890895                                                                     ArgAspArgMetHisHisAspIleValArgLeuLeuAspGluTyrAsn                              900905910                                                                     ValThrProSerProProGlyThrValLeuThrSerAlaLeuSerPro                              915920925                                                                     ValIleCysGlyProAsnArgSerPheLeuSerLeuLysHisThrPro                              930935940                                                                     MetGlyLysLysSerArgArgProSerAlaLysSerThrMetProThr                              945950955960                                                                  SerLeuProAsnLeuAlaLysGluAlaLysAspAlaLysGlySerArg                              965970975                                                                     ArgLysLysSerLeuSerGluLysValGlnLeuSerGluSerSerVal                              980985990                                                                     ThrLeuSerProValAspSerLeuGluSerProHisThrTyrValSer                              99510001005                                                                   AspThrThrSerSerProMet                                                         10101015                                                                      (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1068 amino acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Human N1 (TAN-1)                                                (B) LOCATION: 1152...2219                                                     (D) OTHER INFORMATION: Highly conserved ankyrin repeat                        region of Notch                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       ProSerProCysGlnAsnGlyAlaThrCysThrAspTyrLeuGlyGly                              151015                                                                        TyrSerCysLysCysValAlaGlyTyrHisGlyValAsnCysSerGlu                              202530                                                                        GluIleAspGluCysLeuSerHisProCysGlnAsnGlyGlyThrCys                              354045                                                                        LeuAspLeuProAsnThrTyrLysCysSerCysProTrpGlyThrGln                              505560                                                                        GlyValHisCysGluIleAsnValAspAspCysAsnProProValAsp                              65707580                                                                      ProValSerTrpSerProLysCysPheAsnAsnGlyThrCysValAsp                              859095                                                                        GlnValGlyGlyTyrSerCysThrCysProProGlyPheValGlyGlu                              100105110                                                                     ArgCysGluGlyAspValAsnGluCysLeuSerAsnProCysAspAla                              115120125                                                                     ArgGlyThrGlnAsnCysValGlnArgValAsnAspPheHisCysGlu                              130135140                                                                     CysArgAlaGlyHisThrGlyArgArgCysGluSerValIleAsnGly                              145150155160                                                                  CysLysGlyLysProCysLysAsnGlyGlyThrCysAlaValAlaSer                              165170175                                                                     AsnThrAlaArgGlyPheIleCysLysCysProAlaGlyPheGluGly                              180185190                                                                     AlaThrCysGluAsnAspAlaArgThrCysGlySerLeuArgCysLeu                              195200205                                                                     AsnGlyGlyThrCysIleSerGlyProArgSerProThrCysLeuCys                              210215220                                                                     LeuGlyProPheThrGlyProGluCysGlnPheProAlaSerSerPro                              225230235240                                                                  CysLeuGlyGlyAsnProCysTyrAsnGlnGlyThrCysGluProThr                              245250255                                                                     SerGluSerProPheTyrArgCysLeuCysProAlaLysPheAsnGly                              260265270                                                                     LeuLeuCysHisIleLeuAspTyrSerPheGlyGlyGlyAlaGlyArg                              275280285                                                                     AspIleProProProLeuIleGluGluAlaCysGluLeuProGluCys                              290295300                                                                     GlnGluAspAlaGlyAsnLysValCysSerLeuGlnCysAsnAsnHis                              305310315320                                                                  AlaCysGlyTrpAspGlyGlyAspCysSerLeuAsnPheAsnAspPro                              325330335                                                                     TrpLysAsnCysThrGlnSerLeuGlnCysTrpLysTyrPheSerAsp                              340345350                                                                     GlyHisCysAspSerGlnCysAsnSerAlaGlyCysLeuPheAspGly                              355360365                                                                     PheAspCysGlnArgAlaGluGlyGlnCysAsnProLeuTyrAspGln                              370375380                                                                     TyrCysLysAspHisPheSerAspGlyHisCysAspGlnGlyCysAsn                              385390395400                                                                  SerAlaGluCysGluTrpAspGlyLeuAspCysAlaGluHisValPro                              405410415                                                                     GluArgLeuAlaAlaGlyThrLeuValValValValLeuMetProPro                              420425430                                                                     GluGlnLeuArgAsnSerSerPheHisPheLeuTrpGluLeuSerArg                              435440445                                                                     ValLeuHisThrAsnValValPheLysArgAspAlaHisGlyGlnGln                              450455460                                                                     MetIlePheProTyrTyrGlyArgGluGluGluLeuArgLysHisPro                              465470475480                                                                  IleLysArgAlaAlaGluGlyTrpAlaAlaProAspAlaLeuLeuGly                              485490495                                                                     GlnValLysAlaSerLeuLeuProGlyGlySerGluGlyGlyTrpTrp                              500505510                                                                     TrpArgGluLeuAspProMetAspValArgGlySerIleValTyrLeu                              515520525                                                                     GluIleAspAsnTrpGlnCysValGlnAlaSerSerGlnCysPheGln                              530535540                                                                     SerAlaThrAspValAlaAlaPheLeuGlyAlaLeuAlaSerLeuGly                              545550555560                                                                  SerLeuAsnIleProTyrLysIleGluAlaValGlnSerGluThrVal                              565570575                                                                     GluProProProProAlaGlnLeuHisPheMetTyrValAlaAlaAla                              580585590                                                                     AlaPheValLeuLeuPhePheValGlyCysGlyValLeuLeuSerArg                              595600605                                                                     LysArgTrpXaaGlnHisGlyGlnLeuTrpPheProGluGlyPheLys                              610615620                                                                     ValSerGluAlaSerLysLysLysTrpTrpGluXaaLeuGlyGluAsp                              625630635640                                                                  SerValGlyLeuLysProLeuLysAsnAlaSerAspGlyAlaLeuMet                              645650655                                                                     AspAspAsnGlnAsnGluTrpGlyAspGluAspLeuGluThrLysLys                              660665670                                                                     PheTrpPheGluGluProValValLeuProAspLeuAspAspGlnThr                              675680685                                                                     AspHisTrpGlnTrpThrGlnGlnHisLeuAspAlaAlaAspLeuArg                              690695700                                                                     MetSerAlaMetAlaProThrProProGlnGlyGluValAspAlaAsp                              705710715720                                                                  CysMetAspValAsnValArgGlyProAspGlyPheThrProLeuMet                              725730735                                                                     IleAlaSerCysSerGlyGlyGlyLeuGluThrGlyAsnSerGluGlu                              740745750                                                                     GluGluAspAlaProAlaValIleSerAspPheIleTyrGlnGlyAla                              755760765                                                                     SerLeuHisAsnGlnThrAspArgThrGlyGluThrAlaLeuHisLeu                              770775780                                                                     AlaAlaArgTyrSerArgSerAspAlaAlaLysArgLeuLeuGluAla                              785790795800                                                                  SerAlaAspAlaAsnIleGlnAspAsnMetGlyArgThrProLeuHis                              805810815                                                                     AlaAlaValSerAlaAspAlaGlnGlyValPheGlnIleLeuIleTrp                              820825830                                                                     AsnArgAlaThrAspLeuAspAlaArgMetHisAspGlyThrThrPro                              835840845                                                                     LeuIleLeuAlaAlaArgLeuAlaValGluGlyMetLeuGluAspLeu                              850855860                                                                     IleAsnSerHisAlaAspValAsnAlaValAspAspLeuGlyLysSer                              865870875880                                                                  AlaLeuHisTrpAlaAlaAlaValAsnAsnValAspAlaAlaValVal                              885890895                                                                     LeuLeuLysAsnGlyAlaAsnLysAspMetGlnAsnAsnArgGluGlu                              900905910                                                                     ThrProLeuPheLeuAlaAlaTrpGluGlySerTyrGluThrAlaLys                              915920925                                                                     ValLeuLeuAspHisPheAlaAsnTrpAspIleThrAspHisMetAsp                              930935940                                                                     ArgLeuProArgAspIleAlaGlnGluArgMetHisHisAspIleVal                              945950955960                                                                  ArgLeuLeuAspGluTyrAsnLeuValArgSerProGlnLeuHisGly                              965970975                                                                     AlaProLeuGlyGlyThrProThrLeuSerProProLeuCysSerPro                              980985990                                                                     AsnGlyTyrLeuGlySerLeuLysProGlyValGlnGlyLysLysVal                              99510001005                                                                   ArgLysProSerSerLysGlyLeuAlaCysGlySerLysGluAlaLys                              101010151020                                                                  AspLeuLysAlaTrpArgLysLysSerGlnAspGlyLysGlyCysLeu                              025103010351040                                                               LeuAspSerSerGlyMetLeuSerProValAspSerLeuGluSerPro                              104510501055                                                                  HisGlyTyrLeuSerAspValAlaSerProProLeu                                          10601065                                                                      (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1064 amino acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: unknown4                                                        (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Xen N                                                           (B) LOCATION: 1150...2213                                                     (D) OTHER INFORMATION: Highly conserved ankyrin repeat                        region of Notch                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ProAsnProCysGlnAsnGlyAlaThrCysThrAspTyrLeuGlyGly                              151015                                                                        TyrSerCysGluCysValAlaGlyTyrHisGlyValAsnCysSerGlu                              202530                                                                        GluIleAsnGluCysLeuSerHisProCysGlnAsnGlyGlyThrCys                              354045                                                                        IleAspLeuIleAsnThrTyrLysCysSerCysProArgGlyThrGln                              505560                                                                        GlyValHisCysGluIleAsnValAspAspCysThrProPheTyrAsp                              65707580                                                                      SerPheThrLeuGluProLysCysPheAsnAsnGlyLysCysIleAsp                              859095                                                                        ArgValGlyGlyTyrAsnCysIleCysProProGlyPheValGlyGlu                              100105110                                                                     ArgCysGluGlyAspValAsnGluCysLeuSerAsnProCysAspSer                              115120125                                                                     ArgGlyThrGlnAsnCysIleGlnLeuValAsnAspTyrArgCysGlu                              130135140                                                                     CysArgGlnGlyPheThrGlyArgArgCysGluSerValValAspGly                              145150155160                                                                  CysLysGlyMetProCysArgAsnGlyGlyThrCysAlaValAlaSer                              165170175                                                                     AsnThrGluArgGlyPheIleCysLysCysProProGlyPheAspGly                              180185190                                                                     AlaThrCysGluTyrAspSerArgThrCysSerAsnLeuArgCysGln                              195200205                                                                     AsnGlyGlyThrCysIleSerValLeuThrSerSerLysCysValCys                              210215220                                                                     SerGluGlyTyrThrGlyAlaThrCysGlnTyrProValIleSerPro                              225230235240                                                                  CysAlaSerHisProCysTyrAsnGlyGlyThrCysGlnPhePheAla                              245250255                                                                     GluGluProPhePheGlnCysPheCysProLysAsnPheAsnGlyLeu                              260265270                                                                     PheCysHisIleLeuAspTyrGluPheProGlyGlyLeuGlyLysAsn                              275280285                                                                     IleThrProProAspAsnAspAspIleCysGluAsnGluGlnCysSer                              290295300                                                                     GluLeuAlaAspAsnLysValCysAsnAlaAsnCysAsnAsnHisAla                              305310315320                                                                  CysGlyTrpAspGlyGlyAspCysSerLeuAsnPheAsnAspProTrp                              325330335                                                                     LysAsnCysThrGlnSerLeuGlnCysTrpLysTyrPheAsnAspGly                              340345350                                                                     LysCysAspSerGlnCysAsnAsnThrGlyCysLeuTyrAspGlyPhe                              355360365                                                                     AspCysGlnLysValGluValGlnCysAsnProLeuTyrAspGlnTyr                              370375380                                                                     CysLysAspHisPheGlnAspGlyHisCysAspGlnGlyCysAsnAsn                              385390395400                                                                  AlaGluCysGluTrpAspGlyLeuAspCysAlaAsnMetProGluAsn                              405410415                                                                     LeuAlaGluGlyThrLeuValLeuValValLeuMetProProGluArg                              420425430                                                                     LeuLysAsnAsnSerValAsnPheLeuArgGluLeuSerArgValLeu                              435440445                                                                     HisThrAsnValValPheLysLysAspSerLysGlyGluTyrLysIle                              450455460                                                                     TyrProTyrTyrGlyAsnGluGluGluLeuLysLysHisHisIleLys                              465470475480                                                                  ArgSerThrAspTyrTrpSerAspAlaProSerAlaIlePheSerThr                              485490495                                                                     MetLysGluSerIleLeuLeuGlyArgHisArgArgGluLeuAspGlu                              500505510                                                                     MetGluValArgGlySerIleValTyrLeuGluIleAspAsnArgGln                              515520525                                                                     CysTyrLysSerSerSerGlnCysPheAsnSerAlaThrAspValAla                              530535540                                                                     AlaPheLeuGlyAlaLeuAlaSerLeuGlySerLeuAspThrLeuSer                              545550555560                                                                  TyrLysIleGluAlaValLysSerGluAsnMetGluThrProLysPro                              565570575                                                                     SerThrLeuTyrProMetLeuSerMetLeuValIleProLeuLeuIle                              580585590                                                                     IlePheValPheMetMetValIleValAsnLysLysArgArgArgGlu                              595600605                                                                     HisAspSerPheGlySerProThrAlaLeuPheGlnLysAsnProAla                              610615620                                                                     LysArgAsnGlyGluThrProTrpGluAspSerValGlyLeuLysPro                              625630635640                                                                  IleLysAsnMetThrAspGlySerPheMetAspAspAsnGlnAsnGlu                              645650655                                                                     TrpGlyAspGluGluThrLeuGluAsnLysArgPheArgPheGluGlu                              660665670                                                                     GlnValIleLeuProGluLeuValAspAspLysThrAspProArgGln                              675680685                                                                     TrpThrArgGlnHisLeuAspAlaAlaAspLeuArgIleSerSerMet                              690695700                                                                     AlaProThrProProGlnGlyGluIleGluAlaAspCysMetAspVal                              705710715720                                                                  AsnValArgGlyProAspGlyPheThrProLeuMetIleAlaSerCys                              725730735                                                                     SerGlyGlyGlyLeuGluThrGlyAsnSerGluGluGluGluAspAla                              740745750                                                                     SerAlaAsnMetIleSerAspPheIleGlyGlnGlyAlaGlnLeuHis                              755760765                                                                     AsnGlnThrAspArgThrGlyGluThrAlaLeuHisLeuAlaAlaArg                              770775780                                                                     TyrAlaArgAlaAspAlaAlaLysArgLeuLeuGluSerSerAlaAsp                              785790795800                                                                  AlaAsnValGlnAspAsnMetGlyArgThrProLeuHisAlaAlaVal                              805810815                                                                     AlaAlaAspAlaGlnGlyValPheGlnIleLeuIleArgAsnArgAla                              820825830                                                                     ThrAspLeuAspAlaArgMetPheAspGlyThrThrProLeuIleLeu                              835840845                                                                     AlaAlaArgLeuAlaValGluGlyMetValGluGluLeuIleAsnAla                              850855860                                                                     HisAlaAspValAsnAlaValAspGluPheGlyLysSerAlaLeuHis                              865870875880                                                                  TrpAlaAlaAlaValAsnAsnValAspAlaAlaAlaValLeuLeuLys                              885890895                                                                     AsnSerAlaAsnLysAspMetGlnAsnAsnLysGluGluThrSerLeu                              900905910                                                                     PheLeuAlaAlaArgGluGlySerTyrGluThrAlaLysValLeuLeu                              915920925                                                                     AspHisTyrAlaAsnArgAspIleThrAspHisMetAspArgLeuPro                              930935940                                                                     ArgAspIleAlaGlnGluArgMetHisHisAspIleValHisLeuLeu                              945950955960                                                                  AspGluTyrAsnLeuValLysSerProThrLeuHisAsnGlyProLeu                              965970975                                                                     GlyAlaThrThrLeuSerProProIleCysSerProAsnGlyTyrMet                              980985990                                                                     GlyAsnMetLysProSerValGlnSerLysLysAlaArgLysProSer                              99510001005                                                                   IleLysGlyAsnGlyCysLysGluAlaLysGluLeuLysAlaArgArg                              101010151020                                                                  LysLysSerGlnAspGlyLysThrThrLeuLeuAspSerGlySerSer                              025103010351040                                                               GlyValLeuSerProValAspSerLeuGluSerThrHisGlyTyrLeu                              104510501055                                                                  SerAspValSerSerProProLeu                                                      1060                                                                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1139 amino acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Dros N                                                          (B) LOCATION: 1189...2327                                                     (D) OTHER INFORMATION: Highly conserved ankyrin repeat                        region of Notch                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       SerGlnProCysGlnAsnGlyGlyThrCysArgAspLeuIleGlyAla                              151015                                                                        TyrGluCysGlnCysArgGlnGlyPheGlnGlyGlnAsnCysGluLeu                              202530                                                                        AsnIleAspAspCysAlaProAsnProCysGlnAsnGlyGlyThrCys                              354045                                                                        HisAspArgValMetAsnPheSerCysSerCysProProGlyThrMet                              505560                                                                        GlyIleIleCysGluIleAsnLysAspAspCysLysProGlyAlaCys                              65707580                                                                      HisAsnAsnGlySerCysIleAspArgValGlyGlyPheGluCysVal                              859095                                                                        CysGlnProGlyPheValGlyAlaArgCysGluGlyAspIleAsnGlu                              100105110                                                                     CysLeuSerAsnProCysSerAsnAlaGlyThrLeuAspCysValGln                              115120125                                                                     LeuValAsnAsnTyrHisCysAsnCysArgProGlyHisMetGlyArg                              130135140                                                                     HisCysGluHisLysValAspPheCysAlaGlnSerProCysGlnAsn                              145150155160                                                                  GlyGlyAsnCysAsnIleArgGlnSerGlyHisHisCysIleCysAsn                              165170175                                                                     AsnGlyPheTyrGlyLysAsnCysGluLeuSerGlyGlnAspCysAsp                              180185190                                                                     SerAsnProCysArgValGlyAsnCysValValAlaAspGluGlyPhe                              195200205                                                                     GlyTyrArgCysGluCysProArgGlyThrLeuGlyGluHisCysGlu                              210215220                                                                     IleAspThrLeuAspGluCysSerProAsnProCysAlaGlnGlyAla                              225230235240                                                                  AlaCysGluAspLeuLeuGlyAspTyrGluCysLeuCysProSerLys                              245250255                                                                     TrpLysGlyLysArgCysAspIleTyrAspAlaAsnTyrProGlyTrp                              260265270                                                                     AsnGlyGlySerGlySerGlyAsnAspArgTyrAlaAlaAspLeuGlu                              275280285                                                                     GlnGlnArgAlaMetCysAspLysArgGlyCysThrGluLysGlnGly                              290295300                                                                     AsnGlyIleCysAspSerAspCysAsnThrTyrAlaCysAsnPheAsp                              305310315320                                                                  GlyAsnAspCysSerLeuGlyIleAsnProTrpAlaAsnCysThrAla                              325330335                                                                     AsnGluCysTrpAsnLysPheLysAsnGlyLysCysAsnGluGluCys                              340345350                                                                     AsnAsnAlaAlaCysHisTyrAspGlyHisAspCysGluArgLysLeu                              355360365                                                                     LysSerCysAspThrLeuPheAspAlaTyrCysGlnLysHisTyrGly                              370375380                                                                     AspGlyPheCysAspTyrGlyCysAsnAsnAlaGluCysSerTrpAsp                              385390395400                                                                  GlyLeuAspCysGluAsnLysThrGlnSerProValLeuAlaGluGly                              405410415                                                                     AlaMetSerValValMetLeuMetAsnValGluAlaPheArgGluIle                              420425430                                                                     GlnAlaGlnPheLeuArgAsnMetSerHisMetLeuArgThrThrVal                              435440445                                                                     ArgLeuLysLysAspAlaLeuGlyHisAspIleIleIleAsnTrpLys                              450455460                                                                     AspAsnValArgValProGluIleGluAspThrAspPheAlaArgLys                              465470475480                                                                  AsnLysIleLeuTyrThrGlnGlnValHisGlnThrGlyIleGlnIle                              485490495                                                                     TyrLeuGluIleAspAsnArgLysCysThrGluCysPheThrHisAla                              500505510                                                                     ValGluAlaAlaGluPheLeuAlaAlaThrAlaAlaLysHisGlnLeu                              515520525                                                                     ArgAsnAspPheGlnIleHisSerValArgGlyIleLysAsnProGly                              530535540                                                                     AspGluAspAsnGlyGluProProAlaAsnValLysTyrValIleThr                              545550555560                                                                  GlyIleIleLeuValIleIleAlaLeuAlaPhePheGlyMetValLeu                              565570575                                                                     SerThrGlnArgLysArgAlaHisGlyValThrTrpPheProGluGly                              580585590                                                                     PheArgAlaProAlaAlaValMetSerArgArgArgArgAspProHis                              595600605                                                                     GlyGlnGluMetArgAsnLeuAsnLysGlnValAlaMetGlnSerGln                              610615620                                                                     GlyValGlyGlnProGlyAlaHisTrpSerAspAspGluSerAspMet                              625630635640                                                                  ProLeuProLysArgGlnArgSerAspProValSerGlyValGlyLeu                              645650655                                                                     GlyAsnAsnGlyGlyTyrAlaSerAspHisThrMetValSerGluTyr                              660665670                                                                     GluGluAlaAspGlnArgValTrpSerGlnAlaHisLeuAspValVal                              675680685                                                                     AspValArgAlaIleMetThrProProAlaHisGlnAspGlyGlyLys                              690695700                                                                     HisAspValAspAlaArgGlyProCysGlyLeuThrProLeuMetIle                              705710715720                                                                  AlaAlaValArgGlyGlyGlyLeuAspThrGlyGluAspIleGluAsn                              725730735                                                                     AsnGluAspSerThrAlaGlnValIleSerAspLeuLeuAlaGlnGly                              740745750                                                                     AlaGluLeuAsnAlaThrMetAspLysThrGlyGluThrSerLeuHis                              755760765                                                                     LeuAlaAlaArgPheAlaArgAlaAspAlaAlaLysArgLeuPheHis                              770775780                                                                     AlaGlyAlaAspAlaAsnCysGlnAspAsnThrGlyArgThrProLeu                              785790795800                                                                  HisAlaAlaValAlaAlaAspAlaMetGlyValPheGlnIleLeuLeu                              805810815                                                                     ArgAsnArgAlaThrAsnLeuAsnAlaArgMetHisAspGlyThrThr                              820825830                                                                     ProLeuIleLeuAlaAlaArgLeuAlaIleGluGlyMetValGluAsp                              835840845                                                                     LeuIleThrAlaAspAlaAspIleAsnAlaAlaAspAsnSerGlyLys                              850855860                                                                     ThrAlaLeuHisTrpAlaAlaAlaValAsnAsnThrGluAlaValAsn                              865870875880                                                                  IleLeuLeuMetHisHisAlaAsnArgAspAlaGlnAspAspLysAsp                              885890895                                                                     GluThrProLeuPheLeuAlaAlaArgGluGlySerTyrGluAlaCys                              900905910                                                                     LysAlaLeuLeuAspAsnPheAlaAsnArgGluIleThrAspHisMet                              915920925                                                                     AspArgLeuProArgAspValAlaSerGluArgLeuHisHisAspIle                              930935940                                                                     ValArgLeuLeuAspGluHisValProArgSerProGlnMetLeuSer                              945950955960                                                                  MetThrProGlnAlaMetIleGlySerProProProGlyGlnGlnGln                              965970975                                                                     ProGlnLeuIleThrGlnProThrValIleSerAlaGlyAsnGlyGly                              980985990                                                                     AsnAsnGlyAsnGlyAsnAlaSerGlyLysGlnSerAsnGlnThrAla                              99510001005                                                                   LysGlnLysAlaAlaLysLysAlaLysLeuIleGluGlySerProAsp                              101010151020                                                                  AsnGlyLeuAspAlaThrGlySerLeuArgArgLysAlaSerSerLys                              025103010351040                                                               LysThrSerAlaAlaSerLysLysAlaAlaAsnLeuAsnGlyLeuAsn                              104510501055                                                                  ProGlyGlnLeuThrGlyGlyValSerGlyValProGlyValProPro                              106010651070                                                                  ThrAsnSerAlaValGlnAlaAlaAlaAlaAlaAlaAlaAlaValAla                              107510801085                                                                  AlaMetSerHisGluLeuGluGlySerProValGlyValGlyMetGly                              109010951100                                                                  GlyAsnLeuProSerProTyrAspThrSerSerMetTyrSerAsnAla                              105111011151120                                                               MetAlaAlaProLeuAlaAsnGlyAsnProAsnThrGlyAlaLysGln                              112511301135                                                                  ProProSer                                                                     __________________________________________________________________________

What is claimed is:
 1. A method for expansion of a human precursor cellcomprising contacting the cell in vitro with an amount of an agonist ofNotch function effective to inhibit differentiation of the cell, andexposing the cell in vitro to cell growth conditions such that the cellproliferates.
 2. The method according to claim 1 wherein the precursorcell is of ectodermal origin.
 3. The method according to claim 1 whereinthe precursor cell is of endodermal origin.
 4. The method according toclaim 1 wherein the precursor cell is of mesodermal origin.
 5. Themethod according to claim 1 wherein the precursor cell is selected fromthe group consisting of hematopoietic precursor cells, epithelialprecursor cells, kidney precursor cells, neural precursor cells, skinprecursor cells, osteoblast precursor cells, chondrocyte precursorcells, and liver precursor cells.
 6. The method according to claim 1wherein the agonist is a Delta protein or a derivative thereof whichbinds to Notch.
 7. The method according to claim 1 wherein the agonistis a Serrate protein or a derivative thereof which binds to Notch. 8.The method according to claim 1 wherein the agonist is an antibody to aNotch protein or a fragment of the antibody containing a binding regionthereof.
 9. The method according to claim 1 wherein the precursor cellis a hematopoietic stem cell.
 10. The method according to claim 1wherein the precursor cell contains a recombinant nucleic acid encodinga protein of value in the treatment of a human disease or disorder. 11.The method according to claim 1 wherein the agonist is a Delta orSerrate protein and said contacting is carried out by a methodcomprising exposing the precursor cell to cells recombinantly expressingthe agonist.
 12. The method according to claim 1 wherein said contactingis carried out by culturing said precursor cell in medium containing apurified agonist in soluble form.
 13. The method according to claim 1wherein substantially no differentiation of the cell occurs.
 14. Themethod according to claim 1 in which said contacting and exposing stepsare carried out concurrently.
 15. A method for expansion of a precursorcell comprising contacting the cell in vitro with an amount of a solubleagonist of Notch function effective to inhibit differentiation of thecell, and exposing the cell in vitro to cell growth conditions such thatthe cell proliferates.
 16. The method according to claim 15 wherein theprecursor cell is selected from the group consisting of hematopoieticprecursor cells, epithelial precursor cells, kidney precursor cells,neural precursor cells, skin precursor cells, osteoblast precursorcells, chondrocyte precursor cells, liver precursor cells, and musclecells.
 17. The method according to claim 15 wherein the precursor cellis a hematopoietic stem cell.
 18. The method according to claim 15wherein substantially no differentiation of the cell occurs.
 19. Themethod according to claim 15 wherein the soluble agonist is a derivativeof a Delta protein, which derivative binds to a Notch protein.
 20. Themethod according to claim 19 wherein the derivative of Delta consistsessentially of the extracellular domain of a Delta protein.
 21. Themethod according to claim 19 wherein the derivative is a fragment of aDelta protein.
 22. The method according to claim 15 wherein the solubleagonist is a derivative of a Serrate protein, which derivative binds toa Notch protein.
 23. The method according to claim 22 wherein thederivative of Serrate consists essentially of the extracellular domainof a Serrate protein.
 24. The method according to claim 22 wherein thederivative is a fragment of a Serrate protein.
 25. The method accordingto claim 15 wherein the soluble agonist is an antibody to a Notchprotein or a fragment of the antibody containing a binding regionthereof.
 26. The method according to claim 15 wherein the precursor cellcontains a recombinant nucleic acid encoding a protein of value in thetreatment of a disease or disorder.
 27. The method according to claim 15which further comprises after said contacting step the step ofintroducing into the cell a recombinant nucleic acid encoding a proteinof value in the treatment of a disease or disorder.
 28. The methodaccording to claim 1 or 15 which further comprises removing the agonistof Notch function and inducing at least some of the resulting expandedcells to differentiate.
 29. A method for expansion of a precursor cellcomprising recombinantly expressing within the cell an amount of aDeltex protein or fragment thereof which binds to a Notch proteineffective to inhibit differentiation of the cells; and exposing the cellin vitro to cell growth conditions such that the cell proliferates. 30.A method for expansion of a hematopoietic precursor cell comprisingrecombinantly expressing within the cell an amount of a Notch proteinconsisting essentially of the intracellular domain of a Notch proteineffective to inhibit differentiation; and exposing the cell in vitro tocell growth conditions such that the cell proliferates.
 31. A method forexpansion of an epithelial precursor cell comprising recombinantlyexpressing within the cell an amount of a Notch protein consistingessentially of the intracellular domain of a Notch protein effective toinhibit differentiation; and exposing the cell in vitro to cell growthconditions such that the cell proliferates.
 32. A method for expansionof a liver precursor cell comprising recombinantly expressing within thecell an amount of a Notch protein consisting essentially of theintracellular domain of a Notch protein effective to inhibitdifferentiation; and exposing the cell in vitro to cell growthconditions such that the cell proliferates.
 33. A method for expansionof a human precursor cell comprising contacting the precursor cell invitro with a second cell wherein the second cell recombinantly expresseson its surface a molecule consisting of at least the extracellulardomain of a Notch ligand; and exposing the precursor cell in vitro tocell growth conditions such that the precursor cell proliferates. 34.The method of claim 33 wherein the second cell recombinantly expresseson its surface at least the extracellular domain of a Delta protein. 35.The method of claim 33 wherein the second cell recombinantly expresseson its surface at least the extracellular domain of a Serrate protein.36. A method for expansion of an hematopoietic precursor cell comprisingcontacting the precursor cell in vitro with a second cell wherein thesecond cell recombinantly expresses on its surface a molecule consistingof at least the extracellular domain of a Notch ligand; and exposing theprecursor cell in vitro to cell growth conditions such that theprecursor cell proliferates.
 37. A method for expansion of a humanprecursor cell comprising contacting the precursor cell in vitro with anamount of a second cell expressing a Notch ligand effective to inhibitdifferentiation of the cell; and exposing the precursor cell in vitro tocell growth conditions such that the precursor cell proliferates.
 38. Amethod for promoting mammalian neuronal cell growth comprisingcontacting a mammalian neuron in vitro with an antagonist of Notchfunction and exposing the neuron in vitro to neuronal cell growthconditions.
 39. A method for expansion of a precursor cell comprisingcontacting the cell in vitro with an amount of a soluble fragment of aDelta protein effective to inhibit differentiation of the cell, andexposing the cell in vitro to cell growth conditions such that the cellproliferates.
 40. A method for expansion of a precursor cell comprisingcontacting the cell in vitro with an amount of a soluble fragment of aSerrate protein effective to inhibit differentiation of the cell, andexposing the cell in vitro to cell growth conditions such that the cellproliferates.